Dominance of community-associated methicillin-resistant Staphylococcus aureus clones in a maternity hospital
Dominance of community-associated methicillin-resistant Staphylococcus aureus clones in a maternity hospital
Edet E. Udo 0 1
Noura Al-Sweih 0 1
0 Department of Microbiology, Faculty of Medicine, Kuwait University , Jabriya , Kuwait
1 Editor: Karsten Becker , Universitatsklinikum Munster , GERMANY
Methicillin- resistant Staphylococcus aureus (MRSA) is a major pathogen causing healthcare- and community- acquired infections. The purpose of this study was to characterize MRSA isolated at the Maternity Hospital between 2006 and 2011 for their genetic relatedness.
Data Availability Statement: All relevant data are
within the paper.
Funding: This study was funded by Research
Sector, Kuwait University, grant MI 01/11.
Competing interests: The authors have declared
that no competing interests exist.
The study revealed a high prevalence of community-associated MRSA Maternity hospital.
The MRSA population consisted of known strains, such as ST239-III-t680, ST22-IV-t223/ t852 and ST80-IV-t044, that were reported previously in Kuwait and novel strains such as
ST5-V-t002, and several sporadic strains obtained for the first time in the Maternity hospital.
This study has provided an initial data which will serve as a platform for future comparative studies on the distribution of MRSA clones in the Maternity hospital in Kuwait.
Methicillin-resistant Staphylococcus aureus (MRSA) is a major cause of healthcare- as well as
community-acquired infections [
Methicillin resistance is determined by the production of an altered low affinity
penicillinbinding protein (PBP) called PBP2a (or PBP2') [
]. PBP2a is encoded by the mecA gene,
which confers resistance to all beta-lactam antibiotics and is located on a mobile genetic island
called staphylococcal cassette chromosome mec (SCCmec). In addition, to mecA, SCCmec
contains site-specific cassette chromosome recombinases (ccr) that is responsible for the
integration of the SCCmec into the S. aureus genome. The combination of SCCmecÐand
recombinase gene complexes defines SCCmec types, which differ in size and structural
]. The differences in SCCmec types have been used to type MRSA for
epidemiological purposes and to distinguish healthcare-associated MRSA (HA-MRSA) from
communityassociated MRSA (CA-MRSA). Genetically, CA-MRSA harbor the staphylococcal cassette
chromosome mec (SCCmec) type IV or type V [
], VI or VII [
] whereas HA-MRSA carries
SCCmec types I, II and III [
With the advent of CA-MRSA, infections caused by MRSA have increased in the
community settings globally [
]. In addition, the prevalence of MRSA infections and colonization
among pregnant women have increased [
8, 9, 10
]. Similarly, the number of reports linking
MRSA with outbreaks in newborn nurseries and neonatal intensive care units are also
11, 12, 13, 14, 15
The Maternity hospital in Kuwait is a 500-bed tertiary hospital which handles 12000±14000
deliveries, accounting for approximately 30% of all deliveries, in the State of Kuwait [
MRSA was not reported in the Maternity Hospital in Kuwait prior to 2005 [
]. This was
attributed to its select patient's population. It was argued that pregnant women admitted to the
hospital for child delivery were usually not sick, and spent few days in the hospital after
delivery thereby eliminating the risk factor for the acquisition and transmission of healthcareÐ
associated MRSA. However, the situation has since changed and the number of MRSA isolated
at the Maternity hospital has been increasing gradually following the isolation of a single
MRSA strain in 2005. Molecular typing of 22 MRSA isolates recovered from babies in the
Special babies care unit of the Maternity hospital between 1 October and 31 December 2011,
revealed the presence of CA-MRSA clones that were different from those usually isolated at
other hospitals in Kuwait [
]. This finding revealed a lack of appreciation of the prevailing
MRSA clones and their resistance profiles in other facilities of the hospital and demanded
further investigations of MRSA isolated previously in the hospital. Consequently, to fully
appreciate the epidemiology of MRSA in the Maternity hospital, MRSA that were isolated between
2006 and 2011 were characterized using a combination of molecular typing methods.
Knowledge of the clonal types and their resistance patterns would provide useful background data
which will be useful in assessing future changes in the MRSA population as well as assessing
effectiveness of infection prevention and control measures designed to prevent MRSA
becoming endemic in the hospital.
2 / 12
Materials and methods
The MRSA isolates were obtained as part of routine diagnostic microbiology testing and later
submitted to the MRSA Reference Laboratory for molecular typing. In total, 103 MRSA
isolates recovered between 2006 and 2011 from 64 neonates (72 isolates), 17 adult patients (18
isolates) and 12 healthcare workers (13 isolates) were investigated. The MRSA isolates were
from 37 skin and soft tissues samples, 13 umbilical cord stumps, 18 nasal swabs, 11 eye swabs,
7 blood samples, 4 Vaginal swabs, and 13 miscellaneous sources. The MRSA isolates were
identified using standard bacteriological methods including growth on Mannitol Salt Agar,
Gram stain, tube coagulase and DNase testing and preserved in 40% glycerol (v/v) in nutrient
broth at -80ÊC.
Antibiotic susceptibility testing
Susceptibility to benzyl penicillin, cefoxitin, kanamycin, gentamicin, erythromycin,
clindamycin, chloramphenicol, tetracycline, minocycline, trimethoprim, fusidic acid, rifampicin,
ciprofloxacin, mupirocin and linezolid was determined by the disk diffusion method [
Susceptibility to fusidic acid was interpreted following the British Society for antimicrobial
chemotherapy guidelines [
]. Minimum inhibitory concentration (MIC) was determined for
cefoxitin, mupirocin, vancomycin, and teicoplanin with Etest strips (BioMerieux, Marcey l'
Etoile, France) and interpreted following guidelines by the Clinical and Laboratory Standards
]. MIC for vancomycin was performed by the Etest macro method per the
manufacturer's protocol. S. aureus strain ATCC25923 was used as quality control strain for
susceptibility testing. Methicillin resistance was confirmed by detecting PBP 2a in culture supernatants
using a rapid latex agglutination kit (Denka-Seiken, Japan) following the manufacturer's
instruction. Multiple resistance was defined as resistance to three or more classes of
Detection of genes coding for Panton-Valentine leukocidin, (PVL)
The LukS-PV- lukF-PV gene which codes for PVL was amplified as described previously [
SCCmec typing and subtyping
SCCmec typing was performed by PCR assays as described previously [
Spa typing was performed as described by Harmsen et al. [
] for all MRSA isolates. DNA
sequencing was performed using a 3130x1 genetic analyzer (Applied Bio systems, Forster City,
CA. USA) in accordance with the manufacturer protocol. Isolates were assigned to spa types
using the spa typing website (http://www.spaserver.ridom.de) .
Multilocus sequence typing (MLST)
MLST was performed on all 37 isolates as described by Enright et al., . Isolates were
assigned a sequence type (ST) according to the MLST website (http://www.mlst.net) .
Clonal complexes were defined as group of sequence types (STs) that share at least five of
seven identical alleles with at least one of the STs in the group [
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All MRSA isolates were susceptible to vancomycin, (MIC 2.0 mg/L), teicoplanin
(MIC 2.0 mg/L), linezolid and rifampicin but were resistant to penicillin G (N = 93; 90.3%),
gentamicin (N = 42; 40.8%), kanamycin (N = 48; 46.6%), trimethoprim (N = 33; 32.0%),
ciprofloxacin (N = 23; 22.3%), fusidic acid (N = 17;16.5%), tetracycline (N = 20;19.4%),
erythromycin (N = 16;15.5%), clindamycin (N = 16;15.5%), streptomycin (N = 12;11.6%), high-level
mupirocin (N = 3; 2.9%) and chloramphenicol (N = 1; 0.9%).
Thirty-one (30.0%) isolates obtained from skin and soft tissue infections (13 isolates), nasal
swabs (2 isolates), groin (2 isolates), HVS (3 isolates), and one each from blood, eye swab,
catheter tip and unspecified source were positive for genes encoding PVL.
Molecular typing of MRSA isolates
Molecular typing yielded three SCCmec types (III, IV and V), 28 spa types, 19 sequence types
(ST), 23 clones and 11 clonal complexes (CC) (Table 1). Fifty-seven (55.3%) of the isolates
carried SCCmec IV genetic element, followed by SCCmec V (40 isolates, 38.8%) and SCCmec III
(6 isolates, 5.8%). SCCmec types I and II were not detected.
The major spa types were t002 (N = 24), t223 (N = 18), t852 (N = 10), t044 (N = 7), t304
(N = 7), t688 (N = 6), t657 (N = 5), t860 (N = 4), t1234 (N = 2), t16202 (N = 2), t359 (N = 2)
and t690 (N = 2). Spa types, t005, t019, t037, t105, t15316, t16184, t2051, t267, t311, t314, t318,
t985, t376, t6728 occurred in single isolates. ST5 (N = 31) and ST22 (N = 29) were the
dominant sequence types with both accounting for 58.2% of the isolates. Other common STs were
ST80 (N = 8; 7.7%), ST932 (N = 7; 6.8%), ST772 (N = 5; 4.8%), ST239 (N = 5; 4.8%) and ST97
(N = 4; 3.8%).
The dominant clonal complexes were CC5 (N = 37), CC22 (N = 30), CC80 (N = 8), CC6
(N = 7), CC8 (N = 6) and CC1 (N = 5).
CC1 consisted of five ST772-V-t657 isolates. Four of these isolates were positive for PVL
and were resistant to gentamicin, kanamycin, and trimethoprim. One isolate was negative for
PVL and was susceptible to the non-beta-lactam antibiotics tested. The isolates were obtained
from three babies (3 isolates) and adult patients (2 isolates) (Table 2).
CC5 comprised diverse clones with ST5-V-t002 as the dominant clone, followed by
ST5-Vt688. The ST5-V-t002 isolates were negative for PVL, but were resistant to gentamicin and
kanamycin and variable resistance to other antibiotics (Table 1). ST5-V-t002 was widely
distributed and was isolated from babies, adult patients (Table 2) and healthcare workers
(Table 3). All five ST5-V-t688 isolates were resistant to tetracycline but expressed variable
resistance to erythromycin, clindamycin and chloramphenicol. ST5-V-t688 isolates were
recovered from babies, adult patients (Table 2) and healthcare workers (Table 3).
CC6 consisted of seven ST932-IV-t304 isolates that were negative for PVL and uniformly
susceptible to the non-beta-lactam antibiotics tested. The isolates were obtained from babies,
one healthcare worker and three adult patients including a mother and her baby (Table 2).
CC8 consisted of six isolates that belonged to ST239-III-t860 (4 isolates), ST239-III-t16202
(1 isolate) and ST241-III-t037 (1 isolate). The CC8 isolates were negative for PVL and
expressed multi antibiotic resistance. The t860 isolates were resistant to high-level mupirocin.
ST239-III isolates were recovered only from babies while the ST241-III-t037 isolates was
obtained from an adult patient (Table 2).
CC22 consisted of ST22- MRSA isolates carrying SCCmec IV genetic elements, and
belonged to four different spa types, t005, t223, t15316 and t852 with t223, detected in 18
4 / 12
isolates, as the dominant spa type. The ST22 isolates differed in the carriage of genes for PVL
and antibiotic resistance (Table 1). The ST22-IV-t005 and most of the ST22-IV-t852 isolates
expressed multi antibiotic resistance whereas the ST22-IVa-t223 isolates were susceptible to
most of the non-beta-lactam antibiotics. Whereas the t005 and t852 isolates were positive for
PVL and negative for toxic shock syndrome toxin gene, the t223 isolates were negative for PVL
but positive for toxic shock syndrome toxin gene. The ST22-IVa-t223 isolates were obtained
from colonization as well as infection sites of babies, adult patients (Table 2) and healthcare
workers (Table 3). The t852 isolates were isolated from babies and a baby cot.
CC30 was represented by three single clones, ST30-IVa-t318, ST77-IV-t019 and
ST1055-IV-t2051 that were positive for PVL and susceptible to most non-beta-lactam
CC80 consisted of ST80-IV-t044 (7 isolates) that were positive for PVL and variably
resistant to kanamycin, tetracycline, fusidic acid, trimethoprim, erythromycin and clindamycin,
5 / 12
*; identical to isolates from their babies.
NS; not speci®ed; HVS, high vaginal swab.
and ST80-IV-t376 (1isolate) that was negative for PVL and resistant to erythromycin and
clindamycin only. ST80-IV-t044 isolates were obtained from babies, adult patients (Table 2) and
healthcare workers (Table 3). CC9, CC88 and CC121 consisted of single isolates while CC97
consisted of four isolates comprising two ST97-V-t1234 and ST97-V-t359 (Table 1).
Distribution of MRSA isolates by year
The isolates were analyzed by year of isolation to ascertain if any of the clones persisted in any
units of the hospital. Fig 1 presents the MRSA isolates by year of isolation. Number of isolates
6 / 12
Fig 1. Distribution of MRSA clones by year: 2006±2011.
increased from two in 2006 to a peak of 43 in 2008 with most clones occurring sporadically.
The dominant clones, ST5-V-t002 and ST22-IVa-t223, were isolated in multiple years in
clusters. Fifteen of the 22 ST5-V-t002 isolates were obtained in 2008. Eleven of these isolates were
obtained from babies and a mother in the same ward depicting local transmission. The
remaining seven isolates were obtained in 2010 (6 isolates) and 2011 (1 isolate) from four
different wards. The six ST5-V-t002 isolates obtained in 2010 were all from babies in one ward.
ST5-V-t688 isolates were obtained in 2007 (2 isolates), 2008 (2 isolates), and 2010 (1 isolate).
The ST22-IV isolates, obtained in 2007, 2008, 2010 and 2011, carried different spa types.
Most (9/18) of the ST22-IVa-t223 isolates were obtained in 2007 while four and three of the
isolates were obtained in 2008 and 2011 respectively. Six of the 10 ST22-IV-t852 isolates were
recovered in 2010 from five babies and a baby cot in the same ward. The other three isolates
were obtained in 2011. The ST80-IV-t044 isolates were all obtained in 2007 (2 isolates), 2008
(4 isolates) and 2011 (1 isolate).
ST932-IV-t304 were isolated in 2007 (2 isolates), 2008 (1 isolate) and 2011 (4 isolates). The
ST772-V-t657 were isolated in 2008 (3 isolates) and 2011 (2 isolates).
As the epidemiology of MRSA continue to change, the emergence of novel clones with novel
virulence factors necessitates regular surveillance for better infection control and management
of infection. Molecular typing revealed that the MRSA population in the Maternity hospital
belonged to diverse genetic backgrounds dominated by internationally recognized clonal
complexes CC5, CC22, CC80, CC6, CC8, CC1 and CC97. The major clones were ST5-V-t002
(21.3%), ST22-IVa-t223 (16.5%), ST22-IV-t852 (9.7%), ST932-IV-t304 (6.7%), ST80-IV- t044/
t376 (7.7%), ST772-V-657 (4.8%) and ST239-III-t860 (3.8%). Apart from ST239-III/ST241-III
and ST22-IV, which are healthcare- associated MRSA clones, the majority (66.0%) of the
isolates had community-associated MRSA (CA-MRSA) genotypes. The high prevalence of
CA-MRSA observed in this study mirrors recent observations in other centers where
CA-MRSA are the dominant MRSA isolates obtained from neonatal units [
12, 28, 29, 30,
This study also revealed that the MRSA population consisted of strains that have been
reported previously in other hospitals in Kuwait, and those reported for the first time at the
Maternity hospital. Isolates reported previously at other hospitals include ST239-III-t860,
ST241-III-t037, ST5-IV-t688, ST22-IV-t223/t852, ST80-IV-t044/t376, ST772-V-657,
ST121-V7 / 12
t314, ST5-IV-t002 and ST97-V-t1234 [
]. Unexpectedly, ST239-III-MRSA, which is the
dominant MRSA clone encountered in other Kuwait hospitals , was present in small
numbers in this study. Secondly, the ST239-III-t860 isolates, obtained only in 2007, did not persist
in the Maternity hospital in contrast to their persistence in other hospitals in Kuwait [
Most of the ST22-IVa-t223 isolates were obtained from samples of skin and soft tissue
infections as well colonization sites as was the case in other hospitals in Kuwait  supporting
their ability to be involved in different types of infections. In contrast, the UK-EMRSA-15/
Barnim epidemic MRSA clone is mostly associated with bloodstream infections [
detection of the tst-positive ST22-IVa-t223 isolates among different patients' populations in
this hospital suggests that it is becoming an important epidemic healthcare- associated
pathogen in Kuwait hospitals. In addition to its growing prominence in Kuwait hospitals, tst-positive
ST22-IVa-t223 isolates have also been reported widely in the Gaza strip [
], Jordan [
Saudi Arabia and Egypt [
] and is becoming the leading variant of the UK Epidemic
MRSA15 clone in the Middle East affecting adults and babies. Besides the tst-positive ST22-IVa-t223
isolates, PVL-positive -ST22-IV-t852 MRSA also contributed to the MRSA burden among the
neonates. In addition, a t16202 variant of ST22-IV-MRSA was also detected for the first time
in this study adding to the ongoing diversification of CC22 strains observed in recent years
36, 41, 42
The isolation of ST80-IV-MRSA in eight (7.7%) of the isolates in this study is comparable
to the prevalence of 7.5% prevalence that was reported recently in other hospitals in Kuwait
]. However, with only two of the eight isolates obtained from neonates, the results suggest
that ST80-IV-MRSA had limited role in infections among the neonates. In contrast,
ST80-IVt044 clone was recently reported as the leading cause of infections among neonates in a
hospital in Algeria [
Two of the four ST97-V-MRSA isolate, ST97-V-t359 isolates, were resistant to gentamicin,
kanamycin and fusidic acid like the resistance profiles of ST97-V-MRSA isolates that caused
an outbreak in a neonatal intensive care unit in another Kuwait hospital in 2007 [
other two ST97-V-t1234 isolates were resistant to tetracycline and resembled isolates reported
previously in another Kuwait hospital [
]. ST97-V-MRSA isolates have also been reported
sporadically from Jordan [
], Saudi Arabia [
], Lebanon [
] and countries outside the
Middle East including Australia [
], USA [
], UK [
], and Brazil [
] underlying their growing
importance as a human pathogen in addition to their roles as veterinary pathogens [
Novel MRSA clones isolated at the Maternity hospital included the dominant clone,
ST5-V-t002, recovered from healthcare workers, adult patients and neonates, and sporadic
clones such as ST77-IV-t019, and ST9-V-t267. Although newly isolated in Kuwait, ST5-V-t002
isolates have been reported to cause infections in other countries [
Prior to the report of the ST9-V-t267 isolate in this study, CC9 isolates were recovered from
animals or farm workers [
]. The present isolate was obtained from a blood sample of a baby
admitted to the Special Care unit. However, history of parental contact with animals could not
The isolation of the common MRSA clones, ST5-V-t002, ST22-IVa-t223 and ST80-IV-t044
from neonates, healthcare workers and adult patients in the hospital is suggestive of
nosocomial transmission of these clones. The presence of colonized healthcare workers, neonates and
their mothers, and adult patients in other wards of the hospital have previously been
documented to provide opportunities for nosocomial transmissions of MRSA in a neonatal ward
]. Furthermore, the isolation of ST5-V-t002 from a mother, her baby and nine other babies
in the same unit in 2008 is suggestive of an outbreak with the mother as the likely source of the
]. Likewise, the detection of ST22-IV-t852 from a baby's cot and five different
babies in the same unit suggests that the environment also contributed to the nosocomial
8 / 12
]. Therefore, multiple sources including colonized mothers, adult patients,
healthcare workers and the environment contributed to the MRSA population in the
The study further sought to establish whether any of the clones persisted in the hospital
overtime. The results indicated that although ST5-V-t002, ST22-IVa-t223 and ST80-IV-t044
were isolated in more than one year, the numbers isolated per year were small which suggested
multiple acquisition rather than persistence. The other isolates occurred sporadically
supporting their transient acquisition.
Although the isolates were susceptible to vancomycin, teicoplanin, linezolid and rifampicin,
their susceptibility patterns followed predictable patterns with HA-MRSA isolates, ST239/
ST241 ±III-MRSA, ST22-IV-t005 and ST22-IV-t852 MRSA expressing multi resistance
whereas the CA-MRSA isolates belonging to CC6 (ST6-IV-t6269, ST932-IV-t304 and
ST932-IV-t657) were susceptible to all non-beta-lactam antibiotics and the other CA-MRSA
clones were susceptible to most of the non- beta-lactam antibiotics.
In conclusion, the MRSA isolates belonged to diverse genetic backgrounds with
ST5-Vt002 and ST22-IVa-t223 as the dominant clones. Multiple sources including healthcare
workers, colonized or infected patients, colonized mothers and the environment contributed to the
MRSA population which consisted of MRSA clones that have been isolated widely in other
Kuwait hospitals and sporadic novel clones. This study has provided an initial data which will
serve as a platform for future comparative studies on the distribution of MRSA clones in the
Maternity hospital in Kuwait.
Conceptualization: EEU NA.
Formal analysis: EEU NA.
Funding acquisition: EEU.
Investigation: EEU NA.
Project administration: EEU.
Resources: EEU NA.
Supervision: EEU NA.
Validation: EEU NA.
Visualization: EEU NA.
Writing ± original draft: EEU.
Writing ± review & editing: EEU NA.
9 / 12
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