Dominance of community-associated methicillin-resistant Staphylococcus aureus clones in a maternity hospital

Background 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. Materials and methods The MRSA isolates were investigated using a combination of antibiogram, Staphylococcal chromosome cassette mec (SCCmec) and spa typing to determine their relatedness to MRSA isolated in other Kuwait hospitals. The isolates were also investigated for the carriage of genes for Pantone valentine Leukocidin (PVL). Results A total of 103 MRSA obtained from 64 neonates, 17 adult patients and 12 healthcare workers. The isolates were resistant to Kanamycin (46.6%), gentamicin (40.8%), trimethoprim (32%), ciprofloxacin (22.3%), fusidic acid (16.5%), tetracycline (19.4%), erythromycin (15.5%), clindamycin (15.5%), streptomycin (11.6%) high-level mupirocin (2.9%) and chloramphenicol (0.9%). Twenty (19.4%) of the isolates were multiresistant. Thirty-one (30.0%) isolates were positive for PVL. Molecular typing revealed the presence of 11 clonal complexes and 23 clones with ST5-V-t002, (N = 22), ST22-IV-t223 (N = 18), ST22-IV-t852 (N = 10), ST80-IV-t044 (N = 7), ST5-V-t688 (N = 5), ST772-V-t657 (N = 5) and ST239-III-t860 (N = 4) constituting 66.9% of the isolates. Other clones were isolated sporadically. The number of MRSA isolates increased from two in 2006 to 22 in 2011 with a peak of 43 in 2008. Conclusion 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.


Conclusion
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 PLOS  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.

MRSA isolates
The MRSA isolates were obtained as part of routine diagnostic microbiology testing and later submitted to the MRSA Reference Laboratory for molecular typing.

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 [18]. Susceptibility to fusidic acid was interpreted following the British Society for antimicrobial chemotherapy guidelines [19]. 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 Institute [18]. 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 antibacterial agents.

Detection of genes coding for Panton-Valentine leukocidin, (PVL)
The LukS-PV-lukF-PV gene which codes for PVL was amplified as described previously [20].

Spa typing
Spa typing was performed as described by Harmsen et al. [23] 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) [24].

Multilocus sequence typing (MLST)
MLST was performed on all 37 isolates as described by Enright et al., [25]. Isolates were assigned a sequence type (ST) according to the MLST website (http://www.mlst.net) [26]. 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 [27].
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-V-t688. 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).
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 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.
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-V- t314, ST5-IV-t002 and ST97-V-t1234 [33,34]. Unexpectedly, ST239-III-MRSA, which is the dominant MRSA clone encountered in other Kuwait hospitals [33], 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 [33,35]. 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 [36] 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 [37]. The 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 [38], Jordan [39], Saudi Arabia and Egypt [40] and is becoming the leading variant of the UK Epidemic MRSA-15 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 [33]. 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-IV-t044 clone was recently reported as the leading cause of infections among neonates in a hospital in Algeria [29].
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 [12]. The other two ST97-V-t1234 isolates were resistant to tetracycline and resembled isolates reported previously in another Kuwait hospital [33]. ST97-V-MRSA isolates have also been reported sporadically from Jordan [39], Saudi Arabia [43], Lebanon [44] and countries outside the Middle East including Australia [45], USA [46], UK [47], and Brazil [48] underlying their growing importance as a human pathogen in addition to their roles as veterinary pathogens [49,50].
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 [42,51].
Prior to the report of the ST9-V-t267 isolate in this study, CC9 isolates were recovered from animals or farm workers [42]. 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 be established.
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 [52]. 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 organism [53]. 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 transmission [54]. Therefore, multiple sources including colonized mothers, adult patients, healthcare workers and the environment contributed to the MRSA population in the Maternity hospital.
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.
In conclusion, the MRSA isolates belonged to diverse genetic backgrounds with ST5-V-t002 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.