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Prevalence of methicillin-resistant Staphylococcus aureus (MRSA) infection and the molecular characteristics of MRSA bacteraemia over a two-year period in a tertiary teaching hospital in Malaysia

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Background: Methicillin-resistant Staphylococcus aureus (MRSA) is an established pathogen that causes hospital- and community-acquired infections worldwide. The prevalence rate of MRSA infections were reported to be the highest in Asia. As there is limited epidemiological study being done in Malaysia, this study aimed to determine the prevalence of MRSA infection and the molecular characteristics of MRSA bacteraemia. Methods: Two hundred and nine MRSA strains from year 2011 to 2012 were collected from a tertiary teaching hospital in Malaysia. The strains were characterized by antimicrobial susceptibility testing, staphylococcal cassette chromosome mec (SCCmec) typing, detection of Panton-Valentine leukocidin (PVL) gene, multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE). Patient's demographic and clinical data were collected and correlated with molecular data by statistical analysis. Results: Male gender and patient >50 years of age (p < 0.0001) were significantly associated with the increased risk of MRSA acquisition. Fifty-nine percent of MRSA strains were HA-MRSA that carried SCCmec type II, III, IV and V while 31% were CA-MRSA strains with SCCmec III, IV and V. The prevalence of PVL gene among 2011 MRSA strains was 5.3% and no PVL gene was detected in 2012 MRSA strains. All of the strains were sensitive to vancomycin. However, vancomycin MIC creep phenomenon was demonstrated by the increased number of MRSA strains with MIC ≥1.5 μg/mL (p = 0.008) between 2011 and 2012. Skin disease (p = 0.034) and SCCmec type III (p = 0.0001) were found to be significantly associated with high vancomycin MIC. Forty-four percent of MRSA strains from blood, were further subtyped by MLST and PFGE. Most of the bacteraemia cases were primary bacteraemia and the common comorbidities were diabetes, hypertension and chronic kidney disease. The predominant pulsotype was pulsotype C exhibited by SCCmec III-ST239. This is a first study in Malaysia that reported the occurrence of MRSA clones such as SCCmec V-ST5, untypeable-ST508, SCCmec IV-ST1 and SCCmec IV-ST1137. Conclusions: SCCmec type III remained predominant among the MRSA strains in this hospital. The occurrence of SCCmec IV and V among hospital strains and the presence of SCCmec III in CA-MRSA strains are increasing. MRSA strains causing bacteraemia over the two-year study period were found to be genetically diverse.
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R E S E A R C H A R T I C L E Open Access
Prevalence of methicillin-resistant
Staphylococcus aureus (MRSA) infection and
the molecular characteristics of MRSA
bacteraemia over a two-year period in a
tertiary teaching hospital in Malaysia
Pik San Sit
1
, Cindy Shuan Ju Teh
1
, Nuryana Idris
1
, I-Ching Sam
1
, Sharifah Faridah Syed Omar
2
, Helmi Sulaiman
2
,
Kwai Lin Thong
3
, Adeeba Kamarulzaman
2
and Sasheela Ponnampalavanar
2*
Abstract
Background: Methicillin-resistant Staphylococcus aureus (MRSA) is an established pathogen that causes hospital- and
community-acquired infections worldwide. The prevalence rate of MRSA infections were reported to be the highest in
Asia. As there is limited epidemiological study being done in Malaysia, this study aimed to determine the prevalence of
MRSA infection and the molecular characteristics of MRSA bacteraemia.
Methods: Two hundred and nine MRSA strains from year 2011 to 2012 were collected from a tertiary teaching hospital
in Malaysia. The strains were characterized by antimicrobial susceptibility testing, staphylococcal cassette chromosome
mec (SCCmec) typing, detection of Panton-Valentine leukocidin (PVL) gene, multilocus sequence typing (MLST) and
pulsed-field gel electrophoresis (PFGE). Patients demographic and clinical data were collected and correlated with
molecular data by statistical analysis.
Results: Male gender and patient >50 years of age (p< 0.0001) were significantly associated with the increased risk of
MRSA acquisition. Fifty-nine percent of MRSA strains were HA-MRSA that carried SCCmec type II, III, IV and V while 31%
were CA-MRSA strains with SCCmec III, IV and V. The prevalence of PVL gene among 2011 MRSA strains was 5.3% and
no PVL gene was detected in 2012 MRSA strains. All of the strains were sensitive to vancomycin. However, vancomycin
MIC creep phenomenon was demonstrated by the increased number of MRSA strains with MIC 1.5 μg/mL (p=0.008)
between 2011 and 2012. Skin disease (p= 0.034) and SCCmec type III (p= 0.0001) were found to be significantly
associated with high vancomycin MIC. Forty-four percent of MRSA strains from blood, were further subtyped by
MLST and PFGE. Most of the bacteraemia cases were primary bacteraemia and the common comorbidities were
diabetes, hypertension and chronic kidney disease. The predominant pulsotype was pulsotype C exhibited by
SCCmec III-ST239. This is a first study in Malaysia that reported the occurrence of MRSA clones such as SCCmec
V-ST5, untypeable-ST508, SCCmec IV-ST1 and SCCmec IV-ST1137.
Conclusions: SCCmec type III remained predominant among the MRSA strains in this hospital. The occurrence of
SCCmec IV and V among hospital strains and the presence of SCCmec III in CA-MRSA strains are increasing. MRSA
strains causing bacteraemia over the two-year study period were found to be genetically diverse.
Keywords: Methicillin-resistant Staphylococcus aureus (MRSA), SCCmec types, MLST, PFGE, MRSA Bacteraemia
* Correspondence: sheela@ummc.edu.my
2
Department of Medicine, Faculty of Medicine, University of Malaya, 50603
Kuala Lumpur, Malaysia
Full list of author information is available at the end of the article
© The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and
reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to
the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver
(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
Sit et al. BMC Infectious Diseases (2017) 17:274
DOI 10.1186/s12879-017-2384-y
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
Background
Methicillin-resistant Staphylococcus aureus (MRSA) is
widely recognized as one of the pathogens causing
hospital- and community- acquired infections. MRSA is
highly prevalent in hospitals worldwide in which high
rates (>50%) were reported in Asia, Malta, North and
South America [1]. The prevalence of MRSA in Malaysia
ranged from 17% in 1986 [2] to 44.1% in 2007 [3].
MRSA is evolved from methicillin-susceptible S. aureus
through the acquisition of Staphylococcal cassette
chromosome mec (SCCmec) which carries mecAgene.
mecA gene encodes the penicillin-binding protein (PBP2a)
which confers resistance to all β-lactam antibiotics [4].
Hospital-acquired (HA)-MRSA strains cause nosoco-
mial infections and are associated with SCCmec type I,
II or III. In the 1990s, the epidemiology of MRSA infec-
tions has changed due to the emergence of community-
acquired (CA)-MRSA strains. CA-MRSA strains cause skin
and soft tissue infections (SSTIs), sepsis, osteomyelitis, nec-
rotizing pneumonia and fasciitis and pyomyositis in
children, soldiers, professional football players or incarcer-
ated populations. They often carry Panton-Valentine leuko-
cidin (PVL) genes and SCCmec IV or V [4, 5]. To date,
eleven different SCCmec types (I-XI) have been defined [6].
However, only SCCmec type I-V are globally distributed
while others are uncommon and may exist as local strains
in their original country [7].
There are various discriminative methods to type
MRSA and to determine its molecular epidemiology.
These methods include SCCmec typing, pulsed-field gel
electrophoresis (PFGE) and multilocus sequence typing
(MLST) [1]. In brief, SCCmec polymerase chain reaction
(PCR) typing allows the presumptive assignment of all
SCCmec types to MRSA strains which differentiates
between HA-MRSA and CA-MRSA [8]. PFGE is based
on DNA fingerprints generated by rare restriction endo-
nuclease digestion and is useful for investigating nosoco-
mial outbreaks as well as to identify MRSA strains that
may cause major outbreaks. MLST is a highly discrimin-
atory method used for the investigation of the molecular
evolution of MRSA. It is based on the sequences of
450-bp internal fragments of 7 housekeeping genes
amplified by PCR. The sequences of the genes are com-
pared to those in MLST website (http://www.mlst.net)
which results in an allelic profile or sequence type (ST).
By using the eBURST software (http://eburst.mlst.net/
v3/mlst_datasets/), clonal complex (CC) can be defined
based upon the STs, in which the evolutionary events
can be analyzed [8, 9].
The most prevalent CCs reported worldwide are CC8
(ST239), CC5 (ST5) and CC22 (ST22) [1]. Numerous
genetic linkages of CA-MRSA have been reported to be
globally distributed. These include ST30-IV, ST8-IV,
ST1-IV, ST59-V and ST80-IV [10]. Previous studies done
by Lim et al. [11, 12] on 2003 and 2008 MRSA isolates
in this same tertiary teaching hospital and on 2008 to
2010 MRSA isolates in a tertiary hospital in Terengganu
had reported SCCmec type III and MLST type ST239 to
be the predominant clone. Similar findings were also
reported by Ghaznavi-Rad et al. [13], where 90% of
MRSA infections in another tertiary hospital belonged to
MLST type ST239. CA-MRSA strains in Malaysian
hospitals were reported to belong to SCCmec type
IV-MLST type ST30 based on study done by Ahmad
et al. [3] on 2006 and 2008 MRSA strains from nine
Malaysian hospitals.
Nosocomial bacteraemia has been reported to be
common in hospitals worldwide leading to high mortality
rate [14] and in Malaysia, 21% cases of bacteraemia were
reported to be caused by MRSA [15]. International MRSA
clones such as ST22-SCCmec IV, ST239-SCCmec III,
ST5-SCCmec II [16] ST30-SCCmec IV and ST1-SCCmec
IV [17] were reported to be spreading around the
world, disseminating between and within the coun-
tries. HA-MRSA strains had also been found circulat-
ing in the community and CA-MRSA strains were
reported to cause the outbreak of HA infections [18].
These epidemiological changes in MRSA have posed
great threat to the public health. However, limited
epidemiological studies have been done in Malaysian
hospitals to monitor the trends in MRSA infections.
The importance of continuous surveillance programs
and molecular epidemiological studies; apart from
proper infection control practices and antibiotic
stewardship in designing effective and rational patho-
gen control strategies in hospitals were highlighted in
an example of decreased rate of MRSA infections in
Japan and Taiwan from 71.6% in 2001 to 41% in 2011
and 68.8% in 2000 to 55.9% in 2010, respectively [19]
Therefore, the objectives of the study were to
characterize the MRSA strains from year to 2011 to
2012 and to determine the characteristics of MRSA
bacteraemia in the tertiary teaching hospital by
molecular typing such as SCCmec and PVL PCR
typing, MLST and PFGE.
Methods
Setting
UMMC is a 980-bed referral and teaching hospital in
Malaysia consisting of intensive care units, paediatric,
orthopaedic, surgical, medical, obstetrics and gynaecology
and psychiatry wards and clinics.
Bacterial strains
All adult patients (>16 years old) who fulfilled the
criteria for MRSA infection were included in this study.
This study was approved by Medical Ethic Committee of
University Malaya Medical Centre (UMMC) on 7th June
Sit et al. BMC Infectious Diseases (2017) 17:274 Page 2 of 14
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2014 (MEC ID: 20145-168). A total of 209 non-
duplicate MRSA clinical strains were collected retro-
spectively from University Malaya Medical Centre
(UMMC). The strains were previously isolated from
sterile sites such as cerebrospinal fluid (CSF), synovial
fluid, tissue, bone, pus and blood in January 2011 to
December 2012. Only strains from pus that was
clinically significant and obtained in an aseptic man-
ner either in operation theatre (OT) or procedure
room was included in this study. The identities of the
strains were confirmed with PCR targeting femAgene
and identified as MRSA using standard microbio-
logicalmethodssuchastubecoagulasetest,DNase
and cefoxitin disk (FOX) screen test.
These strains were characterized using antimicrobial
susceptibility testing, SCCmec typing and PCR targeting
PVL gene. Ninety-one selected strains isolated from
blood were further subtyped by PFGE and MLST.
Antimicrobial susceptibility testing
Vancomycin minimum inhibitory concentration (MIC)
results based on E-test were collected from the hospi-
tals microbiology diagnostic laboratory. The interpret-
ation was done according to Clinical and Laboratory
Standards Institute (CLSI) guidelines.
SCCmec typing and PCR-based assays for PVL gene
Crude genomic DNA was extracted by using simple
boiling method and the supernatant was used as DNA
template for PCR analysis. The cycling condition and
primers as described by Milheirico et al. [20] were used
to detect SCCmec types. For further subgrouping
SCCmec type IV strains, the cycling condition as
described by Milheirico et al. [21] and specific primers
4a1, 4a2, 4b1, 4b2, 4c1, 4c2, 4d1 and 4d2 as described by
Zhang et al. [22] were used. The following strains: 85/
2082 (SCCmec type III), JCSC2172 (SCCmec type IVb),
JCSC4744 (SCCmec type IVa), JCSC4469 (SCCmec type
IVd), MR108 (SCCmec type IVc), N315 (SCCmec type II),
NCTC10442 (SCCmec type I) and WIS (SCCmec type V)
were kindly provided by K.L. Thong, University of Malaya
and used as positive control strains to optimize the multi-
plex PCR assay. The detection of PVL gene was
performed as described by Holmes et al. [23] with slight
modification. Briefly, the amplification was performed in a
final volume of 25 μlcontaining2μl of DNA template, 1X
PCR buffer, 1.5 mM MgCI
2
, 0.2 mM dNTPs, 0.5 μlofTaq
polymerase (Promega Corporation, USA) and 0.2 μlof
primers luk-PV-1 and luk-PV-2 (Integrated DNA
Technologies, USA). The cycling conditions were as
described and the PCR products were analyzed by electro-
phoresis in a 1% agarose gel.
MLST and PFGE
MLST was performed as previously described by Enright
et al. [24]. The sequence types (STs) were assigned by
comparing the sequences of each locus to those in the S.
aureus MLST database (http://saureus.mlst.net) and
clonal complexes were defined by eBURST program.
PFGE was performed according to the Centers for
Disease Control and Prevention (CDC) PulseNet proto-
col [25] with slight modification. In brief, a single colony
of bacteria was streaked onto TSA (BD Difco) and
incubated at 37 °C overnight. An aliquot of 100 μlof
bacterial cell suspension (containing bacterial culture in
cell suspension buffer) was transferred to a microcentri-
fuge tube and added with lysostaphin (1 mg/mL) and
lysozyme (10 mg/mL) (Sigma-Aldrich, USA). Following
incubation at 37 °C, proteinase K (20 mg/mL) (Promega
Corporation, USA) and 1% seakem gold agarose (Lonza,
USA) were added into the suspension, mixed and
allowed to solidify in a plug mold. The plug was trans-
ferred into cell lysis buffer (CLB), incubated at 54 °C for
3 h and washed with sterile distilled water and TE buf-
fer. A slice of plug was cut and digested with SmaI (Pro-
mega Corporation, USA) followed by separation on
CHEF MAPPER in 0.5X TBE at 14 °C for 22 h with
pulse time of 5 60 s. The Salmonella ser.Bran-
derup isolate H9812 was used as the reference strain.
The gel was stained in Gel Red dye (Biotium, USA)
and visualized under BioRad GelDoc XR. BioNu-
merics 6.5 Software package was used for cluster ana-
lyses of PFGE profiles based on the unweighted pair
group method with arithmetic averages (UPGMA)
with a tolerance of 1.5% and optimization of 1%. The
PFGE profile was assigned an arbitrary designation
and the Dice coefficient of similarity, Fdefined the
differences [11].
Clinical data collection
Patients demographic and clinical data such as age,
gender, diagnosis, comorbidities, site of infection, health-
care- associated risk factors were collected from the hospi-
tals medical record unit. Based on the CDC definitions,
HA-MRSA is defined as positive culture obtained more
than 48 h after hospital admission, or history of previous
hospitalization or medical procedures. CA-MRSA refers
to cases with positive culture obtained less than 48 h of
admission without healthcare-associated risk factors
[18, 26]. In this study, MRSA infection were defined
as HA- or CA- MRSA based upon the data collected
from patients clinical notes and from the Infection
Control Departments database on multidrug resistant
organisms (MDROs). By using these two sources, the
risk factors for HA- and CA- MRSA infections were
identified with reasonable accuracy.
Sit et al. BMC Infectious Diseases (2017) 17:274 Page 3 of 14
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Table 1 Comparisons between the patientsdemographics, phenotypic and genotypic characteristics of HA-MRSA and CA-MRSA in
2011 and 2012
2011 2012 P
value
Total Pvalue
N= 95 (%) N= 114 (%) N= 209 (%)
Age
50 years old 39 (41.1) 27 (23.7) 0.011 66 (31.6) < 0.0001
> 50 years old 52 (54.7) 86 (75.4) 0.002 138 (66)
Not Known 4 (4.2) 1 (0.9) 5 (2.4)
Gender
Female 30 (31.6) 44 (38.6) 0.312 74 (35.4) < 0.0001
Male 62 (65.3) 70 (61.4) 0.666 132 (63.2)
Not Known 3 (3.2) 0 (0) 3 (1.4)
Source
Blood 40 (42.1) 51 (44.7) 0.780 91 (43.5)
Tissue 37 (38.9) 42 (36.8) 0.776 79 (37.8)
CSF 3 (3.2) 0 (0) 0.092 3 (1.4)
Pus, slough & Abscess 8 (8.4) 11 (9.6) 0.813 19 (9.1)
Pericardial fluid 1 (1.1) 0 (0) 0.455 1 (0.5)
Bullae fluid 0 (0) 1 (0.9) 1.000 1 (0.5)
Synovial fluid 2 (2.1) 2 (1.8) 1.000 4 (1.9)
Bone 4 (4.2) 7 (6.1) 0.758 11 (5.3)
SCCmec types
SCCmec I 0 (0) 0 (0) 0 (0)
SCCmec II 2 (2.1) 0 (0) 0.205 2 (0.9)
SCCmec III 61 (64.2) 78 (68.4) 0.558 139 (66.5)
SCCmec IV
SCCmec IVa 5 (5.3) 4 (3.5) 0.735 9 (4.3)
SCCmec IVb 5 (5.3) 0 (0) 0.018 5 (2.4)
SCCmec IVc 0 (0) 0 (0) 0 (0)
SCCmec IVd 0 (0) 0 (0) 0 (0)
Novel subtypes 15 (15.8) 30 (26.3) 0.090 45 (21.5)
SCCmec V 6 (6.3) 1 (0.9) 0.048 7 (3.3)
Untypeable 1 (1.1) 1 (0.9) 1.000 2 (0.9)
Type of MRSA
HA-MRSA 44 (46.3) 79 (69.3) 0.001 123 (58.9)
SCCmec II 2 (4.5) 0 (0) 0.126 2 (1.6)
SCCmec III 35 (79.5) 56 (70.9) 0.392 91 (73.9)
SCCmec IV
SCCmec IVa 1 (2.3) 3 (3.8) 1.000 4 (3.3)
SCCmec IVb 3 (6.8) 0 (0) 0.044 3 (2.4)
Novel subtypes 3 (6.8) 19 (24.1) 0.025 22 (17.9)
SCCmec V 0 (0) 1 (1.3) 1.000 1 (0.8)
CA-MRSA 41 (43.2) 24 (21.1) 0.001 65 (31.1)
SCCmec III 23 (56.1) 13 (54.2) 1.000 36 (55.4)
SCCmec IV
Sit et al. BMC Infectious Diseases (2017) 17:274 Page 4 of 14
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Statistical analysis
Categorical data were compared using Fisher exact test.
All reported pvalues are two-tailed and analyses were
performed using GraphPad software (https://www.graph
pad.com/quickcalcs/catMenu/). Variables with p<0.05
were considered to be statistically significant.
Results
Distribution of MRSA strains
In this study, 209 MRSA strains from year 2011 to 2012
were collected from various clinical specimens including
tissues (n= 79; 37.8%), blood (n= 91; 43.5%), pus,
slough and abscess (n= 19; 9.1%), cerebrospinal fluid
(n= 3; 1.4%), bone (n= 11; 5.3%), pericardial fluid
(n= 1; 0.5%), bullae fluid (n= 1; 0.5%) and synovial fluid
(n= 4; 1.9%). The median age was 58 years old ranging
from 16 to 92 years old and most cases belonged to the
age group of 5192 years. MRSA infections were signifi-
cantly decreased in the age group of 50 years old
(41.1% in 2011 vs 23.7% in 2012) (p= 0.011) while an
increase was seen in the age group of >50 years old
(54.7% in 2011 vs 75.4% in 2012) (p= 0.002). A total of
132 (63.2%) specimens were collected from male, 74
(35.4%) were from female and 3 specimens were un-
known. Age > 50 years old and male gender (p< 0.0001)
were the significant risk factors in the acquisition of
MRSA.
Fifty-nine percent (123 of 209) of MRSA strains were
HA-MRSA, 31% (65 of 209) were of CA-MRSA and the
remaining MRSA strains were not known. Some of the
MRSA strains had incomplete clinical data such as un-
known age, gender and MRSA types because the
patients medical records could not be retrieved from the
archived records. HA- and CA- MRSA infections were
significantly increased and decreased from year 2011 to
2012 (p= 0.001) respectively (Table 1).
Antimicrobial susceptibility testing
All the MRSA strains were sensitive to vancomycin with
the vancomycin MICs ranged from 0.38 2μg/mL. One
hundred and six (50.7%) and 103 (49.3%) out of 209
strains had vancomycin MIC <1.5 μg/mL and 1.5 μg/mL,
respectively. A significant decreased was observed in the
number of strains with MIC <1.5 μg/mL (61.1% in 2011 vs
42.1% in 2012) and an increase was seen in the number of
strains with MIC 1.5 μg/mL (38.9% in 2011 vs 57.9% in
2012) (p= 0.008). Comparisons of patient demographics,
clinical features and the genotypes between the low
(<1.5 μg/mL) and high (1.5 μg/mL) vancomycin MIC
groups were shown in Table 2. There were no significant
differences in terms of age, gender and clinical diagnosis.
However, skin disease (p=0.034)andSCCmec type
III (p= 0.0001) were found to be strongly associated
with elevated MIC (1.5 μg/mL), while SCCmec
type IV was associated with low vancomycin MIC
(p= 0.0001) (Table 2).
SCCmec types and the presence of PVL gene
For year 2011 MRSA strains, the predominant
SCCmec type was SCCmec type III (n= 61; 64.2%). A
total of 25 MRSA strains were SCCmec type IV and
further subtyped into SCCmec type IVa (n=5;5.3%)
and SCCmec type IVb (n=5;5.3%).FifteenMRSA
strains could not be subtyped and were known as
Table 1 Comparisons between the patientsdemographics, phenotypic and genotypic characteristics of HA-MRSA and CA-MRSA in
2011 and 2012 (Continued)
SCCmec IVa 4 (9.8) 1 (4.2) 0.644 5 (7.7)
SCCmec IVb 2 (4.9) 0 (0) 0.527 2 (3.1)
Novel subtypes 7 (17.1) 10 (41.7) 0.042 17 (26.2)
SCCmec V 4 (9.8) 0 (0) 0.288 4 (6.2)
Untypeable 1 (2.4) 0 (0) 1.000 1 (1.5)
Not Known 10 (10.5) 11 (9.6) 21 (10)
SCCmec III 3 (30) 9 (81.8) 12 (57.1)
SCCmec IV
Novel subtypes 5 (50) 1 (9.1) 6 (28.6)
SCCmec V 2 (20) 0 (0) 2 (9.5)
Untypeable 0 (0) 1 (9.1) 1 (4.8)
PVL gene 5 (5.3) 0 (0) 0.018 5 (2.4)
Vancomycin MIC
< 1.5 μg/mL 58 (61.1) 48 (42.1) 0.008 106 (50.7)
1.5 μg/mL 37 (38.9) 66 (57.9) 103 (49.3)
CSF Cerebrospinal fluid
Fishers exact test done for categorical variables, pvalue < 0.05 was considered to be statistical significant and are indicated by bold text
Sit et al. BMC Infectious Diseases (2017) 17:274 Page 5 of 14
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Table 2 Comparison between the vancomycin MICs with patients demographics, clinical diagnosis and genotypic characteristics of
MRSA in 2011 and 2012
Vancomycin MIC
< 1.5 μg/mL 1.5 μg/mL Pvalue
2011 2012 Total 2011 2012 Total
N= 58 (%) N= 48 (%) N= 106 (%) N= 37 (%) N= 66 (%) N= 103 (%)
Age
50 years old 26 (44.8) 8 (16.7) 34 (32.1) 13 (35.1) 19 (28.8) 32 (31.1) 0.883
> 50 years old 29 (50) 40 (83.3) 69 (65.1) 23 (62.2) 46 (69.7) 69 (66.9) 0.884
Not Known 3 (5.2) 0 (0) 3 (2.8) 1 (2.7) 1 (1.5) 2 (1.9)
Gender
Female 18 (31) 22 (45.8) 40 (37.7) 12 (32.4) 22 (33.3) 34 (33) 0.563
Male 38 (65.5) 26 (54.2) 64 (60.4) 24 (64.9) 44 (66.7) 68 (66) 0.474
Not Known 2 (3.4) 0 (0) 2 (1.9) 1 (2.7) 0 (0) 1 (0.9)
Clinical diagnosis
Bacteraemia 25 (43.1) 24 (50) 49 (46.2) 15 (40.5) 27 (40.9) 42 (40.8) 0.486
Skin and soft tissue infections 26 (44.8) 20 (41.7) 46 (43.4) 20 (54.1) 31 46.9) 51 (49.5) 0.407
Osteomyelitis or septic arthritis 3 (5.2) 3 (6.3) 6 (5.7) 2 (5.4) 7 (10.6) 9 (8.7) 0.432
Meningitis 3 (5.2) 0 (0) 3 (2.8) 0 (0) 1 (1.5) 1 (0.9) 0.622
Pericarditis 1 (1.7) 1 (2.1) 2 (1.9) 0 (0) 0 (0) 0 (0) 0.498
Co-morbidities
Diabetes mellitus 12 (20.7) 27 (56.3) 39 (36.8) 12 (32.4) 25 (37.9) 37 (35.9) 1.000
Hypoglycaemia 1 (1.7) 0 (0) 1 (0.9) 0 (0) 0 (0) 0 (0) 1.000
Hypertension 12 (20.7) 24 (50) 36 (33.9) 11 (29.7) 23 (34.8) 34 (33) 1.000
Obesity 0 (0) 1 (2.1) 1 (0.9) 0 (0) 0 (0) 0 (0) 1.000
Chronic kidney disease and UTI 9 (15.5) 19 (39.6) 28 (26.4) 5 (13.5) 25 (37.9) 30 (29.1) 0.758
Cancer 8 (13.8) 6 (12.5) 14 (13.2) 3 (8.1) 2 (3) 5 (4.9) 0.052
Head injury 3 (5.2) 10 (20.8) 13 (12.3) 6 (16.2) 10 (15.2) 16 (15.5) 0.552
Liver disease 1 (1.7) 5 (10.4) 6 (5.7) 0 (0) 2 (3) 2 (1.9) 0.280
Respiratory disease 7 (12.1) 9 (18.8) 16 (15.1) 3 (8.1) 10 (15.2) 13 (12.6) 0.691
Cardiovascular disease 6 (10.3) 11 (22.9) 17 (16) 2 (5.4) 8 (12.1) 10 (9.7) 0.217
Gastrointestinal disease 2 (3.4) 3 (6.3) 5 (4.7) 0 (0) 2 (3) 2 (1.9) 0.446
Autoimmune disease 1 (1.7) 0 (0) 1 (0.9) 0 (0) 0 (0) 0 (0) 1.000
Bone and joint disorder 3 (5.2) 4 (8.3) 7 (6.6) 0 (0) 6 (9.1) 6 (5.8) 1.000
Endocrine disorder 0 (0) 2 (4.2) 2 (1.9) 0 (0) 1 (1.5) 1 (0.9) 1.000
Blood disorder 1 (1.7) 1 (2.1) 2 (1.9) 0 (0) 2 (3) 2 (1.9) 1.000
CMV 0 (0) 1 (2.1) 1 (0.9) 0 (0) 0 (0) 0 (0) 1.000
Skin disease 0 (0) 1 (2.1) 1 (0.9) 4 (10.8) 3 (4.5) 7 (6.8) 0.034
None 3 (5.2) 4 (8.3) 7 (6.6) 0 (0) 4 (6.1) 4 (3.9) 0.538
Not Known 24 (41.4) 2 (4.2) 26 (24.5) 12 (32.4) 8 (12.1) 20 (19.4) 0.407
SCCmec types
SCCmec II 0 (0) 0 (0) 0 (0) 2 (5.4) 0 (0) 2 (1.9) 0.242
SCCmec III 32 (55.2) 21 (43.8) 53 (50) 29 (78.4) 57 (86.4) 86 (83.5) 0.0001
SCCmec IV
SCCmec IVa 4 (6.9) 3 (6.3) 7 (6.6) 1 (2.7) 1 (1.5) 2 (1.9) 0.171
SCCmec IVb 5 (8.6) 0 (0) 5 (4.7) 0 (0) 0 (0) 0 (0) 0.060
Sit et al. BMC Infectious Diseases (2017) 17:274 Page 6 of 14
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
novel SCCmec type IV subtypes. There were two and
six strains typed as SCCmec type II and SCCmec type V,
respectively. For 2012 MRSA strains, three SCCmec types
were observed: SCCmec type III (n= 78; 68.4%), SCCmec
type IV (n= 34; 29.8%) and SCCmec type V (n=1;0.9%).
The SCCmec type IV strains were further subtyped as
SCCmec type IVa (n=4;3.5%)andnoveltypeIV
SCCmec subtypes (n= 30; 26.3%). There were two
untypeable MRSA strains which harbour mecAgene
and no SCCmec type I was found in both years. PVL
gene was present in five MRSA strains from 2011 but
none was reported in 2012 MRSA strains. All PVL-positive
strains were CA-MRSA strains isolated from pus, tissue
and abscess and carried SCCmec type IV and V. There was
no significant difference in the number of strains carrying
SCCmec types II and III. However, a significant decreased
in the numbers of SCCmec types IVb (p= 0.018) and V
(p= 0.048) from 2011 to 2012 was observed.
Genotyping by PFGE
PFGE of SmaI-digested chromosomal DNA of 91 MRSA
strains generated 8 pulsotypes (A H) each with 13
20 bands, with a Dice coefficient, Franging from 0.5 to
1.0. Based on 80% similarity, three clusters (Cluster I to
Cluster III) and five singletons were observed. The ma-
jority of the strains (64.8%) were clustered in Cluster II,
followed by Cluster I which contained 20 strains (21.9%)
and Cluster III which contained 9 strain (6.6%). Pulso-
type C in Cluster II was the predominant pulsotype seen
with multiple subtypes (C1- C27) and followed by pulso-
type A in Cluster I with subtypes (A1-A12). Pulsotype E
in Cluster III was less common with fewer subtypes (E1-
E4) and pulsotypes B, D, F, G and H had no subtypes.
Most of the MRSA strains belonged to subtype C11,
which was the top major subtype and they were indistin-
guisable or clonally related. The presence of the clonally
or closely related MRSA strains despite being isolated
from different wards and different years may suggest the
persistence of this clone in this hospital and there was
transmission of this clone between wards (Fig. 1).
Determination of clonal relationships by MLST
MLST analysis revealed eight dfferent sequence types
(ST) which include ST239 (n= 61), ST22 (n= 19),
ST508 (n= 1), ST772 (n= 1), ST6 (n= 6), ST5 (n= 1),
ST1 (n= 1) and ST1137 (n= 1). ST6, ST22 and ST239
were observed in MRSA strains of both years, whereas
ST5, ST508 and ST772 were present among the 2011
MRSA strains and ST1 and ST1137 were found in 2012
MRSA strains. SCCmec type III belonged to ST239,
SCCmec type IV belonged to ST1, ST6, ST22 and
ST1137 while SCCmec type V belonged to ST5 and
ST772. Strains with untypeable SCCmec were repre-
sented by two STs (239 and 508). MRSA strains were
clustered into six clonal complexes (CC) based on the
similarity between STs in six of seven loci. ST239 was
assigned as the putative ancestral genotype of a sub-
group within CC8. ST1, ST5, ST6 and ST22 were identi-
fied as the ancestral genotypes of their corresponding
CCs. The newly discovered clones; ST508 and ST1137
were observed in this study and they belonged to CC45
and CC22, respectively (Fig. 2) (Table 3).
Discussion
MRSA is known to be one of the most prominent patho-
gens that causes HA and CA- associated infections in
Malaysian hospitals. In this study, 59% (123 of 209) of
MRSA strains were HA-MRSA and 31% (65 of 209)
were of CA-MRSA. Most of the HA-MRSA infections
were caused by SCCmec type III (n= 91; 73.9%) strains.
Although SCCmec IV and V have been reported to occur
in the community [9], most patients seem to acquire
these strains in this hospital based on the proportions of
SCCmec IV (n= 29; 23.6%) and V (n= 1; 0.8%), indicat-
ing the invasion of these strains into hospitals and may
replace the classical HA-MRSA strains due to their
unique characteristics and faster growth patterns [27].
CA-MRSA infections in this hospital have decreased
from 41 to 24 cases in 2011 and 2012, respectively. The
high prevalence of SCCmec type III (n= 36; 55.4%)
among the CA-MRSA strains might indicate that this
MRSA hospital strain has spread to the community. As
Table 2 Comparison between the vancomycin MICs with patients demographics, clinical diagnosis and genotypic characteristics of
MRSA in 2011 and 2012 (Continued)
Novel subtypes 13 (22.4) 23 (47.9) 36 (33.9) 2 (5.4) 7 (10.6) 9 (8.7) 0.0001
SCCmec V 3 (5.2) 1 (2.1) 4 (3.8) 3 (8.1) 0 (0) 3 (2.9) 1.000
Untypeable 1 (1.7) 0 (0) 1 (0.9) 0 (0) 1 (1.5) 1 (0.9) 1.000
UTI = Urinary Tract Infection, CMV = Cytomegalovirus
Head injury includes: basal ganglia bleed, subdural hematoma and stroke. Liver disease includes: alcoholic liver disease, liver cirrhosis. Respiratory disease includes:
pneumonia, pulmonary embolism, chronic obstructive pulmonary disease, acute respiratory distress syndrome. Cardiovascular disease includes: mitral valve
regurgitation, ischaemic heart disease, acute coronary syndrome, congestive cardiac failure. Gastrointestinal disease includes: acute gastroenteritis, Crohns disease,
perforated diverticular disease, intestinal obstruction. Autoimmune disease includes: Systemic lupus erythematosus. Endocrine disorder includes: thyroid disease,
primary hypothyroidism. Blood disorder includes: myelofibrosis, anaemia. Skin disease includes: Stevens Johnson Syndrome, Bullous pemphigoid, exfoliative
dermatitis and pemphigus vulgaris
Fishers exact test done for categorical variables, pvalue < 0.05 was considered to be statistical significant and are indicated by bold text
Sit et al. BMC Infectious Diseases (2017) 17:274 Page 7 of 14
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Fig. 1 Dendrogram of MRSA PFGE patterns generated by UPGMA clustering method using Dice coefficient. The dotted line indicates an
arbitrary 80% similarity demarcation
Sit et al. BMC Infectious Diseases (2017) 17:274 Page 8 of 14
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
stated by Chen et al. [28] in a recent study in China,
SCCmec III-ST239 is beginning to appear in CA-MRSA
isolates. In addition, in the first international surveillance
study on the CA-MRSA epidemiology in Asian coun-
tries, have revealed the spreading of HA-MRSA isolates
to the community, given the presence of SCCmec types
I, II and III in CA-MRSA isolates from Taiwan, Korea,
Hong Kong, Philippines, Thailand and Vietnam [29].
Based on the distributions analysis in Table 1, MRSA
infections were significantly correlated with male pa-
tients and between the ages of 51 and 92 years.
The existence of the vancomycin creep phenomenon
was demonstrated by the increased percentages of
MRSA strains with MIC 1.5 μg/mL over a 2-year
period. In addition, skin disease and SCCmec type III
were found to be significantly associated with high
vancomycin MIC. This is possibly due to the previous
exposure of MRSA strains to vancomycin which led to
the changes in the characteristics of bacteria such as
bacterial cell wall alterations, preventing vancomycin to
reach its target site [30].
SCCmec type III was the predominant SCCmec type
for year 2011 (n= 61; 64.2%) and 2012 (n= 78; 68.4%)
which is consistent with previous reports in Malaysian
hospitals done by Lim et al. [11] and Ghaznavi-Rad et al.
[13]. SCCmec type III was also common in Asian coun-
tries such as Taiwan, Singapore, Thailand and Indonesia
[19]. For year 2011 MRSA strains, 25 MRSA strains are
SCCmec type IV and were further subtyped as SCCmec
type IVa and IVb. Fifteen SCCmec type IV MRSA strains
could not be further subtyped and might be the novel
type IV SCCmec subtypes. SCCmec types II and V were
also observed. For 2012 MRSA strains, other than
SCCmec type III, SCCmec type IV and V were also
Fig. 2 Population snapshot of MRSA strains from blood source in the MLST database. Individual sequence type (ST) is highlighted in black (STs in
the database), green (STs in this study) and pink (STs in both the database and in this study) and is represented by each circle. The size of the
circle indicates the frequency of a particular ST and the colour of the circle represents founders or subgroup founders. Blue circles are founders
[defined as ST with many single locus variants (SLVs) and is prevalent within a CC], whereas yellow circle are subgroup founders. The most
prevalent ST in this study was ST239, which is the subgroup founder within CC8
Sit et al. BMC Infectious Diseases (2017) 17:274 Page 9 of 14
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
Table 3 Comparison between SCCmec types with the patients demographic, clinical diagnosis and molecular characteristics of
MRSA strains isolated from blood
SCCmec III SCCmec IV SCCmec V Untypeable Total
N= 60 (%) N= 27 (%) N= 2 (%) N= 2 (%) N= 91 (%)
Age
50 years old 12 (20) 3 (11.1) 0 (0) 0 (0) 15 (16.5)
> 50 years old 47 (78.3) 23 (85.2) 2 (100) 2 (100) 74 (81.3)
Not Known 1 (1.7) 1 (3.7) 0 (0) 0 (0) 2 (2.2)
Gender
Female 30 (50) 10 (37) 0 (0) 0 (0) 40 (43.9)
Male 29 (48.3) 16 (59.3) 2 (100) 2 (100) 49 (53.8)
Not Known 1 (1.7) 1 (3.7) 0 (0) 0 (0) 2 (2.2)
Co-morbidities
Diabetes mellitus 20 (33.3) 11 (40.7) 1 (50) 0 (0) 32 (35.2)
Hypertension 22 (36.7) 12 (44.4) 1 (50) 0 (0) 35 (38.5)
Obesity 0 (0) 1 (3.7) 0 (0) 0 (0) 1 (1.1)
Chronic kidney disease and UTI 24 (40) 14 (51.9) 0 (0) 0 (0) 38 (41.8)
Cancer 8 (13.3) 2 (7.4) 0 (0) 0 (0) 10 (10.9)
Head injury 11 (18.3) 4 (14.8) 0 (0) 1 (50) 16 (17.6)
Liver disease 4 (6.7) 2 (7.4) 0 (0) 0 (0) 6 (6.6)
Respiratory disease 12 (20) 3 (11.1) 1 (50) 0 (0) 16 (17.6)
Cardiovascular disease 4 (6.7) 8 (29.6) 2 (100) 0 (0) 14 (15.4)
Gastrointestinal disease 4 (6.7) 1 (3.7) 0 (0) 0 (0) 5 (5.5)
Autoimmune disease 1 (1.7) 0 (0) 0 (0) 0 (0) 1 (1.1)
Bone and joint disorder 1 (1.7) 2 (7.4) 0 (0) 0 (0) 3 (3.3)
Endocrine disorder 0 (0) 2 (7.4) 0 (0) 0 (0) 2 (2.2)
Blood disorder 2 (3.3) 0 (0) 0 (0) 0 (0) 2 (2.2)
CMV 1 (1.7) 0 (0) 0 (0) 0 (0) 1 (1.1)
Skin disease 7 (11.7) 0 (0) 0 (0) 0 (0) 7 (7.7)
None 1 (1.7) 0 (0) 0 (0) 0 (0) 1 (1.1)
Not known 7 (11.7) 5 (18.5) 0 (0) 1 (50) 13 (14.3)
No of comorbidities
0 8 (13.3) 5 (18.5) 0 (0) 1 (50) 14 (15.4)
1 19 (31.7) 5 (18.5) 1 (50) 1 (50) 26 (28.6)
2 11 (18.3) 6 (22.2) 0 (0) 0 (0) 17 (18.7)
3 12 (20) 1 (3.7) 0 (0) 0 (0) 13 (14.3)
4 10 (16.7) 10 (37) 1 (50) 0 (0) 21 (23.1)
Source of bacteraemia
Primary bacteraemia 38 (63.3) 19 (70.4) 2 (100) 2 (100) 61 (67)
Catheter-related 7 (11.7) 5 (18.5) 0 (0) 0 (0) 12 (13.2)
Skin and soft tissue infection 6 (10) 0 (0) 0 (0) 0 (0) 6 (6.6)
Surgical site infection 1 (1.7) 0 (0) 0 (0) 0 (0) 1 (1.1)
Pneumonia 5 (8.3) 2 (7.4) 0 (0) 0 (0) 7 (7.7)
Implant-related infection 0 (0) 1 (3.7) 0 (0) 0 (0) 1 (1.1)
More than one source 3 (5) 0 (0) 0 (0) 0 (0) 3 (3.3)
Sit et al. BMC Infectious Diseases (2017) 17:274 Page 10 of 14
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observed. The SCCmec type IV strains are further sub-
typed as SCCmec type IVa and novel type IV SCCmec
subtypes. There were untypeable MRSA strains which
harbours mecA gene detected in each year. These strains
may indicate new or variant SCCmec types which should
be further analysed [31]. The prevalence of PVL gene
among 2011 MRSA strains was 5.3% and most of them
caused skin and soft tissue infections. No PVL gene was
detected in year 2012 strains.
MRSA bacteraemia is common in hospitals worldwide,
including Malaysia and is associated with high mortality
rate and vancomycin treatment failure. Therefore, se-
lected MRSA strains from blood were further typed by
MLST and PFGE in order to determine the molecular
characteristics of MRSA bacteraemia in both years. In
this study, the MRSA yield from blood for both years
were accounted for 43.5% (n=91)with81.3%(n= 74) re-
corded in patients aged 51 years and more. Most of the
bacteraemia cases were primary bacteraemia (n= 61; 67%).
Diabetes (n= 32; 35.2%), hypertension (n= 35; 38.5%) and
chronic kidney disease (n= 38; 41.8%) were the most
common underlying comorbidities. MRSA bacteraemia
were seen to be caused by HA-MRSA strains type III
(n=36)andIV(n= 15) and CA-MRSA strains type III
(n= 15), IV (n=9)andV(n=2)asshowninTable3.
Analysis of the PFGE patterns have supported the possibil-
ity of the spread of SCCmec type III-ST239 into the com-
munity as they are closely related to the HA-MRSA clone
of SCCmec type III-ST239. Whereas, SCCmec type IV
strains were seen to have invaded and resided in this hos-
pital as nosocomial MRSA strains. Due to their multiplica-
tion and transmission rates, there is a possibility for these
type IV strains to replace and become the predominant
endemic MRSA clone in this hospital [32].
Based on the PFGE dendrogram, 20 MRSA strains
within Cluster I were closely related as they shared more
than 80% similarity. Although they were from different
years, they shared the same characteristics of having
SCCmec type IV and ST22. MRSA strain of SCCmec
type IV-ST1137 was also assigned in this cluster because
Table 3 Comparison between SCCmec types with the patients demographic, clinical diagnosis and molecular characteristics of
MRSA strains isolated from blood (Continued)
Type of MRSA
HA-MRSA 36 (60) 15 (55.6) 0 (0) 0 (0) 51 (56)
CA-MRSA 15 (25) 9 (33.3) 2 (100) 1 (50) 27 (29.7)
Not Known 9 (15) 3 (11.1) 0 (0) 1 (50) 13 (14.3)
CC/ST
CC1
ST1 0 (0) 1 (3.7) 0 (0) 0 (0) 1 (1.1)
ST772 0 (0) 0 (0) 1 (50) 0 (0) 1 (1.1)
CC5
ST5 0 (0) 0 (0) 1 (50) 0 (0) 1 (1.1)
CC6
ST6 0 (0) 6 (22.2) 0 (0) 0 (0) 6 (6.6)
CC8
ST239 60 (100) 0 (0) 0 (0) 1 (50) 61 (67)
CC22
ST22 0 (0) 19 (70.4) 0 (0) 0 (0) 19 (20.9)
ST1137 0 (0) 1 (3.7) 0 (0) 0 (0) 1 (1.1)
CC45
ST508 0 (0) 0 (0) 0 (0) 1 (50) 1 (1.1)
Vancomycin MIC
< 1.5 μg/mL 22 (36.7) 25 (92.6) 1 (50) 1 (50) 49 (53.8)
1.5 μg/mL 38 (63.3) 2 (7.4) 1 (50) 1 (50) 42 (46.2)
UTI = Urinary Tract Infection, CMV = Cytomegalovirus
Head injury includes: basal ganglia bleed, subdural hematoma and stroke. Liver disease includes: alcoholic liver disease, liver cirrhosis. Respiratory disease includes:
pneumonia, pulmonary embolism, chronic obstructive pulmonary disease, acute respiratory distress syndrome. Cardiovascular disease includes: mitral valve
regurgitation, ischaemic heart disease, acute coronary syndrome, congestive cardiac failure. Gastrointestinal disease includes: acute gastroenteritis, Crohns disease,
perforated diverticular disease, intestinal obstruction. Autoimmune disease includes: Systemic lupus erythematosus. Endocrine disorder includes: thyroid disease,
primary hypothyroidism. Blood disorder includes: myelofibrosis, anaemia. Skin disease includes: Stevens Johnson Syndrome, Bullous pemphigoid, exfoliative
dermatitis and pemphigus vulgaris
Sit et al. BMC Infectious Diseases (2017) 17:274 Page 11 of 14
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
ST1137 was shown to belong in the same CC as ST22.
Another six SCCmec type IV MRSA strains were
assigned in Cluster III as they shared similar characteristic
of having MLST ST6. Most novel SCCmec type IV strains
(n= 18) were assigned in the same Cluster I as SCCmec
type IVb. SCCmec type IVa displayed no epidemiological
linkage with SCCmec type IVb. There was a substantial
genetic diversity in SCCmec type IV by the presence of
various sequence types (ST1, ST5, ST6, ST22 and
ST1137) indicating its capability to transfer between
different genetic clones of S. aureus. All the MRSA strains
that possessed SCCmec type III-ST239 were distributed in
Cluster II.
The two SCCmec type V strains found in this study
were CA-MRSA, PVL negative, had low vancomycin
MIC and assigned as singleton due to difference in STs;
ST772 and ST5. Previous studies done in the same
hospital by Lim et al. [11] and Sam et al. [33] had
reported the presence of ST22 and ST6 in SCCmec type
IV and ST772 in SCCmec V. However, no local study
has reported the presence of ST1 and S1137 in SCCmec
type IV strains and ST5 in SCCmec type V strain. Inter-
esting findings were that ST1-SCCmec IV has been
reported by Otter and French in a study done on drug
users and the homeless in South London [34] and
ST5-SCCmec V was found in Japanese CA-MRSA iso-
lates [35] which may indicate the invasion of these
clones into Malaysia. The occurrence of the inter-
national major clones ST239-SCCmec III and ST22-
SCCmec IV in Malaysia as well in other countries
exhibited the epidemic nature of these clones [13].
To date, there is no study reporting ST1137-SCCmec
type IV strains.
The untypeable SCCmec strains with ST239 and
ST508 were not assigned to any clusters and appeared
as singleton by PFGE. The untypeable CA-MRSA
strain with novel ST508 might have derived from
ST45 as it appeared to be a single locus (SLV) variant
of ST45. According to Berglund et al. [36], MRSA
strains that could not be typed by SCCmec typing
might cause by some genetic rearrangement or muta-
tions. This led to the emergence of a new highly
pathogenic CA-MRSA clone which could have spread
to the hospital. ST45 was previously reported as an
epidemic strains in Germany and the Netherlands
[37] and was present in MRSA bacteraemia isolates in
a study done in Switzerland [38].
PFGE was shown to be more discriminatory than
MLST in subtyping the MRSA strains. Based on PFGE,
the strains had greater diversity with Simpsons index of
diversity, D of 0.946 (C.I.N.A. 0.916-0.976), on the other
hand, for MLST, the strains were less diverse with Simp-
sons index of diversity, D of 0.508 (C.I.N.A. 0.403-
0.613). The majority of MRSA strains in this 2-year
study period belonged to the pandemic clone SCCmec
III-ST239 and most of them had pulsotype C. The
predominance of this pulsotype could be due to the
increase in nosocomial transmission within this hos-
pital. In addition, the presence of this clone indicates
the persistence of this clone and was identified as an
endemic strain in this hospital [39].
Limitations
The limitations for this study is the clinical data for
some 2011 and 2012 MRSA strains could not be
retrieved, and hence a comprehensive correlation be-
tween phenotypic, molecular and clinical data cannot
be made.
Conclusions
MRSA strains belonging to SCCmec type III were pre-
dominant in this hospital over the 2-year study period
and the prevalence of PVL gene among 2011 MRSA
strains were 5.3% with none detected in 2012 MRSA
strains. The characteristics of MRSA strains that caused
bacteraemia were genetically diverse by the presence of
different clones circulating in this hospital, in which the
majority belonged to SCCmec type III with MLST
ST239 and exhibited pulsotype C. However, the presence
of SCCmec type IV with MLST ST22 which is usually
community-acquired is gaining prominence as they have
become nosocomial pathogens and may replace the pre-
dominant SCCmec type III strains in this hospital. There
were also emergence of novel clones which require fur-
ther studies and proper monitoring as these clones may
have epidemic and pathogenic potentials that could pose
serious threat to public health.
Abbreviations
CA: Community-acquired; CC: Clonal complex; CDC: Centers for Disease
Control and Prevention; CLB: cell lysis buffer; CLSI: Clinical and Laboratory
Standards Institute; CSB: Cell suspension buffer; CSF: Cerebrospinal fluid;
FOX: Cefoxitin disk; HA: Hospital-acquired; MDROs: Multidrug resistant
Organisms; MIC: Minimum inhibitory concentration; MLST: Multilocus
sequence typing; MRSA: Methicillin-resistant Staphylococcus aureus;
PCR: Polymerase chain reaction; PFGE: Pulsed-field gel electrophoresis;
PVL: Panton-Valentine leukocidin; SCCmec: staphylococcal cassette
chromosome mec; SSTIs: Skin and soft tissue infections; ST: Sequence type;
TE: Tris-EDTA; TSA: Tryptic Soy Agar; UMMC: University Malaya Medical
Centre; UPGMA: Unweighted pair group method with arithmetic averages
Acknowledgements
This research was funded by FRGS, MOE (FP016-2014B) and PPP grant
(PG174-2015B) from Universiti Malaya.
Funding
Not applicable.
Availability of data and materials
All data generated or analyzed during this study are included in this published
article. De-identified raw data will be made available upon reasonable request
addressed to the corresponding author.
Sit et al. BMC Infectious Diseases (2017) 17:274 Page 12 of 14
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
Authorscontributions
PSS carried out the experiments, clinical data collection, data analysis and
interpretation and drafted the manuscript. CSJT participated in study design,
supervised the project, data interpretation and drafted the manuscript. NI
and HS participated in the selection of strains, providing the strains, helped
in the interpretation of antimicrobial susceptibility data and editing of the
manuscript. AK, ICS and SFSO participated in the study design. KLT provided
control strains for SCCmec typing, equipment for PFGE and helped in drafting the
manuscript. SP participated in the study design, coordinated and supervised the
project and co-wrote the manuscript. All authors read and approved the final
manuscript.
Competing interests
The authors declare that they have no competing interests.
Consent for publication
Not applicable.
Ethics approval and consent to participate
This study was approved by Medical Ethics Committee of University Malaya
Medical Centre (UMMC) on 7th June 2014 (MEC ID: 20145-168). This was a
retrospective study and only bacterial strains were collected. No patient was
directly involved, hence obtaining individual patient consent was not required.
PublishersNote
Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations.
Author details
1
Department of Medical Microbiology, Faculty of Medicine, University of
Malaya, 50603 Kuala Lumpur, Malaysia.
2
Department of Medicine, Faculty of
Medicine, University of Malaya, 50603 Kuala Lumpur, Malaysia.
3
Institute of
Biological Sciences, Faculty of Science, University of Malaya, 50603 Kuala
Lumpur, Malaysia.
Received: 29 November 2016 Accepted: 5 April 2017
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... It has been reported that the S. aureus antibiotic susceptibility profiles may vary from region to region [7,11]. The prevalence rate of MRSA infections is increasing in hospitals worldwide with high rates (>50%) reported in Asia, Malta and South America [12][13]. In Malaysia, the prevalence of MRSA among S. aureus isolates ranged from 17% to 35.5% [7,13]. ...
... The prevalence rate of MRSA infections is increasing in hospitals worldwide with high rates (>50%) reported in Asia, Malta and South America [12][13]. In Malaysia, the prevalence of MRSA among S. aureus isolates ranged from 17% to 35.5% [7,13]. Recent Malaysian National Surveillance of Antimicrobial Resistance (NSAR) annual report that included antibiotic susceptibility profiles from the Ministry of Health and universities teaching hospitals in Malaysia revealed that there was an increment of MRSA rate in year 2017 (19.8%) compared to the previous year 2016 (18.0%) [14]. ...
... The MRSA infection was defined as hospital acquired-(HA-) or community acquired-(CA-) MRSA based upon the data collected from the medical record and from the HATTM Infection Control Department's database. Based on the Centres for Disease Control and Prevention (CDC) [13,18], the HA-MRSA is defined as positive culture obtained more than 48 hours after hospital admission, or history of previous hospitalization or medical procedures. Meanwhile, the CA-MRSA refers to cases with positive culture obtained less than 48 hours of admission in the absence of healthcare-associated risk factors [7,13,19]. ...
... It has been reported that the S. aureus antibiotic susceptibility profiles may vary from region to region [7,11]. The prevalence rate of MRSA infections is increasing in hospitals worldwide with high rates (>50%) reported in Asia, Malta and South America [12][13]. In Malaysia, the prevalence of MRSA among S. aureus isolates ranged from 17% to 35.5% [7,13]. ...
... The prevalence rate of MRSA infections is increasing in hospitals worldwide with high rates (>50%) reported in Asia, Malta and South America [12][13]. In Malaysia, the prevalence of MRSA among S. aureus isolates ranged from 17% to 35.5% [7,13]. Recent Malaysian National Surveillance of Antimicrobial Resistance (NSAR) annual report that included antibiotic susceptibility profiles from the Ministry of Health and universities teaching hospitals in Malaysia revealed that there was an increment of MRSA rate in year 2017 (19.8%) compared to the previous year 2016 (18.0%) [14]. ...
... The MRSA infection was defined as hospital acquired-(HA-) or community acquired-(CA-) MRSA based upon the data collected from the medical record and from the HATTM Infection Control Department's database. Based on the Centres for Disease Control and Prevention (CDC) [13,18], the HA-MRSA is defined as positive culture obtained more than 48 hours after hospital admission, or history of previous hospitalization or medical procedures. Meanwhile, the CA-MRSA refers to cases with positive culture obtained less than 48 hours of admission in the absence of healthcare-associated risk factors [7,13,19]. ...
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Background: Staphylococcus aureus is a human pathogen causing a variety of diseases ranging from superficial skin and soft-tissue infections to life-threatening infections. Nowadays, the treatment has become complicated due to the emergence of methicillin-resistant S. aureus (MRSA). It is an established pathogen that causes hospital- and community-acquired infections worldwide. This study was a preliminary retrospective study involving S. aureus clinical isolates from Hospital Angkatan Tentera Tuanku Mizan (HATTM) for a period of 6 months (October 2018 until March 2019). Objectives: The study was conducted to determine the S. aureus antibiotic susceptibility profiles and its association with types of infection, gender, wards, age of patients and types of specimens. Methods: Standard bacteriological method was used for S. aureus isolation from various clinical specimens including blood, urine, pus and wound, eye, throat, nasal, pericardial fluid, tracheal aspirate, high vagina swab (HVS), bronchial alveolar lavage (BAL) and tissue. Kirby-Bauer disc diffusion method was utilized for antibiotic susceptibility profiles determination. Results: Ninety-five S. aureus strains were isolated from 3571 specimens. Twenty-six of the isolates (27%) was identified as MRSA and 69 (73%) was identified as methicillin-sensitive S. aureus (MSSA). Among the MRSA, 4 isolates were hospital-acquired MRSA (HA-MRSA) and the rest were community-acquired MRSA (CA-MRSA). There was a statistically significant association between isolation of MRSA with age of patient as well as types of infection or origin of the MRSA (p<0.05). Only 42 (44.2%) of the S. aureus isolates were fully susceptible to all antibiotics. Almost half isolates (n=46, 48.4%) were resistant to at least two antibiotics and seven (7.4%) isolates were resistant to one type of antibiotic only. Conclusions: None of the isolate showed reduced susceptibility nor resistant to vancomycin. Future study with a larger sample size using a wider study period needs to be done to confirm that vancomycin-resistant S. aureus has not yet been established in HATTM.
... Among MRSA isolates at HCTM, male patients were found to have increased risk of contracting MRSA compared to females, which is in coherence with a 2017 study at another Malaysian tertiary teaching hospital (Sit et al. 2017). Existing literature has shown that the large proportions of risk factors, which predispose individuals to acquire MRSA, were predominantly found in males rather than females. ...
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Methicillin-resistant Staphylococcus aureus (MRSA) is a major nosocomial pathogen that causes severe morbidity, mortality and high medical expenses in many hospitals worldwide. The present study aimed to determine the prevalence of MRSA nosocomial infection, its associated factors, and its antimicrobial susceptibility pattern. This was a retrospective analysis of a database of Multidrug- Resistant Organism (MDRO) that was cultured from patients admitted to Hospital Canselor Tuanku Mukhriz (HCTM) over a period of 2 years (2018-2019). MRSA accounted for 23.6% of total MDRO isolates. The male gender had a higher risk for MRSA acquisition (p<0.05), while the most prevalent setting for MRSA was the orthopaedic ward (47.5%) followed by the medical ward (29.4%). The MRSA strains were significantly isolated from respiratory specimens (55.6%) followed by tissue (50.8%) and blood (27.8%). All MRSA isolates were resistant to penicillin G, oxacillin followed by ciprofloxacin (83.8%) erythromycin (71.5%) and clindamycin (53.5%). MRSA isolates were most susceptible to teicoplanin (99.7%), mupirocin (99.3%), co-trimoxazole (98.4%), rifampicin (97.8%), doxycycline (97.4%), linezolid (95.8%), gentamicin (93.9%) and fusidic acid (86.2%). The trend for MRSA’s antibiotic susceptibility in HCTM for the past 2 years (2018 to 2019) remains unchanged. Further research will be required to investigate the predictor of MRSA by clearly differentiating between MRSA infections and colonisations, hospitalacquired MRSA and community-acquired MRSA.
... Among 27 isolates of S. aureus, 55.6% isolates were screened as methicillin-resistant. Some studies reported similar prevalence of MRSA [28,[46][47][48][49] whereas other studies reported high prevalence of MRSA [19,50]. The variation in the prevalence of MRSA could be due to the difference in locations and time periods of the studies, infection control measures, antibiotic prophylaxis and treatments used in each hospital and the epidemic nature of these microorganisms [51]. ...
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The increasing incidence of methicillin-resistant and biofilm-forming S. aureus isolates in hospital settings is a gruesome concern today. The main objectives of this study were to determine the burden of S. aureus in clinical samples, assess their antibiotic susceptibility pattern and detect biofilm formation and mecA gene in them. A total of 1968 different clinical specimens were processed to isolate S. aureus following standard microbiological procedures. Antibiotic susceptibility test of the isolates was performed by Kirby-Bauer disc-diffusion method following CLSI guidelines. Biofilm was detected through tissue culture plate method. Methicillin-resistant S. aureus (MRSA) isolates were screened using cefoxitin (30 µg) discs and mecA gene was amplified by conventional polymerase chain reaction (PCR). Of 177 bacterial growth, the prevalence of S. aureus was 15.3% (n = 27). MRSA were 55.6% (15/27) and 44% (12/27) exhibited multidrug resistance (MDR). There was no significant association between methicillin resistance and MDR (p > 0.05). Both MRSA and MSSA were least sensitive to penicillin (100%, 75%) followed by erythromycin (86.6%, 66.6%). Most of the MRSA (93.4%) were susceptible to tetracycline. All S. aureus isolates were biofilm producers-19 (70%) were weak and only one (4%) was a strong biofilm producer. The strong biofilm-producing MSSA was resistant to most of the antibiotics except cefoxitin and clindamycin. None of the MSSA possessed mecA gene while 8 (53.3%) MRSA had it. More than half of S. aureus isolated were MRSA. High incidence of multidrug resistance along with capacity to form biofilm among clinical isolates of S. aureus is a matter of apprehension and prompt adoption of biosafety measures is suggested to curb their dissemination in the hospital environments.
... Carriers may transfer the pathogen infecting individuals who may develop disease. In the past few years methicillin resistant S. aureus bacteria has been rapidly emerged due to excessive usage of methicillin within hospitals worldwide (27,22,25). Unambiguous characterization of S. aureus isolates is needed to control MRSA infections through studying the impact of measures and understanding the population biology of S. aureus. ...
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There is so little information available in Iraq about genetic variability in methicillin resistant Staphylococcus aureus (MRSA), so current study aimed to use six tandem repeat loci of multilocus variable number tandem repeat (MLVA) typing to discriminate among MRSA, so to achieve the aim of this study, six loci, clfA, clfB, sdrC, spa, sspa and sav1078 were selected for multiplex PCR. The PCR product were subjected to capillary electrophoresis by using ABI-Genetic analyzer, then the data were analyzed by using GeneMapper™ Software 5. Fragment sizes were converted into repeats number. The total number of repeats are used to generate allelic profile. The allelic profile used to draw the minimum spanning tree and dendrogram , all the 85 MRSA isolates are clustered into 54 MLVA type.
... 4 MRSA evolved from MSSA through SCCmec acquisition which carried mecA gene. 5 This gene encoded PBP2a which has a weaker affinity for β-lactam antibiotic that causes the synthesis of S. aureus peptidoglycan was not interrupted when given an antibiotic. 6,7 MRSA can be classified into HA-MRSA, CA-MRSA and LA-MRSA. ...
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Full-text available
Background: Methicillin-resistant Staphylococcus aureus (MRSA) is penicillin and cephalosporine resistant Staphylococcus aureus which is a major cause of nosocomial infection. Various studies have been conducted for resistant cases, especially herbs which have antibacterial activity. Sambiloto (Andrographis paniculata) is an example of herb which has antibacterial activity due to the presence of andrographolide. Andrographolide soluble in ethanol and poorly in water, while extraction with alcohol causes andrographolide’s degradation. This study aimed to investigate the antimicrobial activity of ethanol and water stem extract and compare them against MRSA ATCC 3351 in vitro.Methods: This is an experimental study with a post-test only control group design and conducted by disc diffusion technique to obtain an inhibition zone against MRSA.Result: The result of this study showed the mean inhibitory zone of ethanol stem extract was 5,87mm, 6,77mm, 7,87mm respectively for 25%, 50%, and 100% concentrations, while 1% concentration did not show antibacterial activity. Water stem extract at 1%, 25%, 50%, and 100% concentrations showed mean inhibitory zone was at 4,57mm, 7,17mm, 8,67mm, and 9,67mm respectively.Conclusion: Ethanol and water stem extract showed antibacterial activity against MRSA. The independent sample t-test didn’t show a difference between ethanol and water stem extract of Andrographis paniculata toward MRSA (p>0,05).
... Hence, we conducted this study to profile antibiotic resistance susceptibility of MSSA and MRSA and type SCCmec elements in MRSA isolates. No MRSA and MSSA isolates were resistant to vancomycin in this study, consistent with the previous studies in Malaysia [29][30][31]. Resistance to penicillin was the highest [7]. All MRSA strains were resistant to penicillin, indicating that they all express PBP2a protein that confers resistance to β-lactam antibiotics. ...
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Full-text available
In general, methicillin-resistant Staphylococcus aureus (MRSA) is more resistant to multiple antibiotics than methicillin-susceptible S. aureus (MSSA). However, some studies have been inconclusive on antibiotic resistance patterns in MSSA and MRSA. This study's objectives were to compare antibiotic resistance patterns of MSSA and MRSA, and to type staphylococcal cassette chromosome mec (SCCmec) in MRSA isolates. S. aureus (MRSA=50; MSSA=50) isolates were collected from the primary teaching hospital in Kuala Lumpur, Malaysia. Antibiotic susceptibility test and amplification of SCCmec elements and antibiotic resistance genes were conducted. MRSA had significantly higher resistance to clindamycin, erythromycin, and ciprofloxacin (P < 0.001) than MSSA, while MSSA had higher resistance to tetracycline than MRSA (P < 0.05). Resistance to gentamicin, fusidic acid, rifampicin, and mupirocin was low in both groups. Multidrug resistance was observed in 14% of MSSA and 66% of MRSA. Remarkably, SCCmec type IV was dominant in MRSA (96%) isolated from this study. All MSSA (5/5) and 27/31 of MRSA harbored ermC, respectively. ermA and AacA-D were not observed in both groups. One MSSA isolate harbored tetK, while tetM was not detected. This study reveals differences in the antibiotic resistance pattern of MSSA and MRSA. The emergence of multidrug resistance in MSSA warrants further surveillance.
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Background: The emergence of multidrug-resistant strains such as methicillin-resistant Staphylococcus aureus (MRSA) and multidrug-resistant Staphylococcus aureus (MDRSA) in animals and humans with continuous contact are a great zoonotic concern. Aims: This cross-sectional study was performed to investigate the carriage rate, genotypic characteristics, and to determine the antibiogram of S. aureus isolated from pets and pet owners in Malaysia. Methods: Nasal and oral swab samples from 40 cats, 30 dogs, and 70 pet owners were collected through convenient sampling. Presumptive colonies on mannitol salt agar were subjected to biochemical identification. S. aureus and MRSA were confirmed by PCR detection of nuc and mecA genes, respectively. Molecular profiles for antimicrobial resistance and virulence genes in S. aureus were also determined. The antibiogram was carried out via Kirby-Bauer test using 18 antibiotics. Results: 17.5% of cats, 20% of dogs, and 27% of pet owners were S. aureus positive. MRSA was also detected in dogs, and pet owners. S. aureus isolates displayed high resistance against penicillin (72.7%), and amoxicillin/clavulanate (66.7%). 39.4% of S. aureus isolates showed multidrug-resistance traits, phenotypically. Molecular characterization of S. aureus revealed the presence of mecA, tetk, tetL, ermA, ermB, ermC, msrA, scn, chp, sak, sep, and sea genes. Conclusion: This study showed the emergence of MRSA and MDRSA in pets and pet owners in Malaysia. The antibiogram findings showed resistance of S. aureus to multiple antibiotics. Furthermore, molecular analysis of immune evasion cluster (IEC) strongly suggests the spread of animal-adapted S. aureus lineages among pets and pet owners.
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Staphylococcus aureus (S. aureus) is an opportunistic pathogen that causes various inflammatory local infections, from those of the skin to postinfectious glomerulonephritis. These infections could result in serious threats, putting the life of the patient in danger. Antibiotic-resistant S. aureus could lead to dramatic increases in human mortality. Antibiotic resistance would explicate the failure of current antibiotic therapies. So, it is obvious that an effective vaccine against S. aureus infections would significantly reduce costs related to care in hospitals. Bacterial vaccines have important impacts on morbidity and mortality caused by several common pathogens, however, a prophylactic vaccine against staphylococci has not yet been produced. During the last decades, the efforts to develop an S. aureus vaccine have faced two major failures in clinical trials. New strategies for vaccine development against S. aureus has supported the use of multiple antigens, the inclusion of adjuvants, and the focus on various virulence mechanisms. We aimed to present a compressive review of different antigens of S. aureus and also to introduce vaccine candidates undergoing clinical trials, from which can help us to choose a suitable and effective candidate for vaccine development against S. aureus. View Full-Text
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Background/purpose: Infections caused by methicillin-resistant Staphylococcus aureus (MRSA) continue to be a problem for clinicians worldwide. The objective of this study was to determine the changes in antibiograms of MRSA and their genotypic characteristics. Methods: The antibiograms of 162 MRSA isolates (52 from 2003 and 110 from 2008) from a tertiary hospital were analyzed by antimicrobial susceptibility tests, the Panton-Valentine leukocidin (PVL) and staphylococcal cassette chromosome mec (SCCmec) types were determined by polymerase chain reaction, and genetic relatedness by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). Results: All the isolates were sensitive to vancomycin. Resistance to ciprofloxacin, clindamycin, erythromycin, and gentamicin remained high throughout the study period, although a small decrease was observed in 2008 for ciprofloxacin (96% to 90%) and gentamicin (90% to 83%). Similarly, a slight decrease in resistance toward fusidic acid (10% to 9%), linezolid (2% to 1%), rifampicin (8% to 4%), and teicoplanin (4% to 0%) was observed between 2003 and 2008. In contrast, there was a significant increase (p < 0.05) in resistance rates toward trimethoprim-sulfamethoxazole, netilmicin, and tetracycline between 2003 and 2008. Ninety-six percent of the isolates from both 2003 and 2008 were multidrug resistant. Three SCCmec types (SCCmec type III, 90%; SCCmec type IV, 9%; SCCmec V, 1%) were observed. SCCmec type IV (n = 15) and pvl gene (n = 3) were detected in 2008 isolates but not in 2003 isolates. Most of the SCCmec type IV isolates (12 of 15) belonged to sequence type 22 (ST22) and were resistant to erythromycin and ciprofloxacin, with 11 being multidrug resistant. Most of the isolates were genetically related (F > 0.8) as determined by PFGE. Some isolates from 6 years apart shared similar PFGE profiles, indicating the persistence of a particular genotype. Five STs (ST239, ST772, ST22, ST6, and ST1178) were identified among the 2008 isolates but only one ST (ST239) was observed in 2003 isolates. Conclusion: Vancomycin remains the most active agent in vitro against S. aureus infection followed by linezolid and teicoplanin. The prevalence of resistance to fluoroquinolones, aminoglycosides (netilmicin), and tetracyclines had increased over the years. The Malaysian multidrug-resistant MRSA isolates were mostly SCCmec type III and ST239, although SCCmec type IV: ST22 is gaining importance. There was a correlation between resistotypes and PFGE profiles.