https://orcid.org/0000-0002-9008-4898
Figures
Abstract
Introduction
Methicillin-resistant Staphylococcus aureus (MRSA) is a highly clonal pathogen causing infections in various settings. The aim of this study was to determine if healthcare-associated (HA) MRSA isolates with the same spa-type originating from two geographically distinct hospitals in South Africa were genetically related based on PFGE. Furthermore, a small subset of MRSA isolates were characterised with WGS and then compared to PFGE to determine if PFGE is still a reliable method to define outbreaks and/or transmission chains.
Methods
Staphylococcus aureus isolated from blood cultures (BC) were submitted to the Centre for Healthcare-Associated Infections, Antimicrobial Resistance and Mycoses (CHARM) as part of a laboratory-based surveillance programme (GERMS-SA). The identified HA-MRSA isolates underwent molecular characterisation [Staphylococcal Chromosome Cassette (SCC) mec and spa-typing]. Pulsed-field gel electrophoresis (PFGE) was performed on selected isolates with the same spa-type. Twenty-one MRSA isolates were selected for whole-genome sequencing (WGS) based on spa-type, PFGE clustering, time and place of isolation.
Results
Eighteen percent (n = 95/529) and 33% (n = 234/710) of isolates collected, from two public tertiary academic hospitals in the Gauteng (GAU) and the Western Cape (WC) provinces, were identified as MRSA, respectively. The most dominant clone in the GAU hospital was t037-III-MRSA (43.2%; n = 41/95). The most dominant clones in the WC hospital was t037-III-MRSA (23.9%, n = 56/234) and t045-I-MRSA (23.5%, n = 55/234). The GAU-t037-III-MRSA cases and WC-t045-I-MRSA cases occurred in the paediatric patient population, whereas the WC-t037-III-MRSA cases occurred in the adult patient population. A novel spa-type (t19935) was detected in the GAU hospital. PFGE showed that the GAU- and WC-t037-III-MRSA isolates were genetically indistinguishable, as well as most of the WC-t045-I-MRSA isolates. The Vienna/Hungarian/Brazilian clone and British EMRSA-3 clone were in circulation and a low frequency of single nucleotide polymorphisms (SNP) (≤20) differences was observed among isolates with the same spa-type.
Conclusion
The low number of SNP differences is suggestive of uninterrupted strain transmission and the persistence of t037-III-MRSA and t045-I-MRSA from 2013 to 2017 in the two studied hospitals. Alternative infection prevention and control strategies should be considered to supplement control efforts.
Citation: Strasheim W, Perovic O, Singh-Moodley A, Kwanda S, Ismail A, Lowe M (2021) Ward-specific clustering of methicillin-resistant Staphylococcus aureus spa-type t037 and t045 in two hospitals in South Africa: 2013 to 2017. PLoS ONE 16(6): e0253883. https://doi.org/10.1371/journal.pone.0253883
Editor: Herminia de Lencastre, The Rockefeller University, UNITED STATES
Received: January 24, 2021; Accepted: June 14, 2021; Published: June 29, 2021
Copyright: © 2021 Strasheim et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the manuscript and its Supporting Information files.
Funding: We know of no conflict of interest associated with this publication and there has been no financial support for this work that could have influenced its outcome.
Competing interests: The authors have declared that no competing interests exist.
Introduction
Methicillin-resistant Staphylococcus aureus (MRSA) is listed as a high priority pathogen by the World Health Organization [1]. This bacterium can cause a wide range of infections but remains an important cause of bacteraemia in both the community and hospital setting [2]. Patients with MRSA bacteraemia pose a burden to any healthcare system, especially those with restrained resources, as these patients are hospitalised for longer, increasing their costs and are reported to have a higher risk of mortality [3,4].
MRSA was first reported in the United Kingdom (UK) in the 1960s [5]. An essential characteristic of MRSA is the presence of the mecA gene, harboured on the Staphylococcal Chromosome Cassette (SCC) mec element, which allows MRSA to survive in the presence of broad-spectrum β-lactams [6]. It was first thought that resistance emerged due to the introduction of methicillin as a therapeutic agent [7]. However, whole-genome sequencing (WGS) analysis of a historical MRSA collection showed that archaic MRSA strains were already in circulation during the 1940s and emerged due to the selective pressure imposed by antibiotic usage combined with poor infection prevention and control measures [7].
S. aureus is a highly clonal pathogen and MRSA isolates with the same Staphylococcal protein A (spa)-type suggest that these isolates were genetically related at some point in time [8,9]. However, inference of strain relatedness from an isolate’s spa-type is problematic, since it involves a single gene; disregards the remainder of S. aureus’ genome and it may miss recombination and homoplasy events [9,10]. Pulsed-field gel electrophoresis (PFGE) has a higher discriminatory power over single gene typing schemes and when combined with epidemiological data, is a valuable tool to confirm the genetic relatedness of MRSA isolates, even though this technique is not considered as the gold standard anymore and is being replaced by WGS [9]. However, WGS is still costly in low- to middle-income countries and it is not financially feasible to sequence large historic collections of isolates.
The first reported South African cases of MRSA was in 1973 in a large paediatric hospital (2%; n = 17/843) [11]. The first hospital outbreak of MRSA in South Africa was in 1986 as nurses noted an increase in infections in the burn, plastic, orthopaedic and trauma wards [12]. Since the first reported MRSA case, an increase in the prevalence of MRSA was observed throughout the country [13–17]. The highest prevalence of MRSA was recorded in 2010 (53%; 297/556), followed by a significant decline (53% to 40%; P ≤0.001) in cases from 2012 to 2016 [17,18]. Currently, the proportion of S. aureus blood culture (BC) isolates resistant to methicillin in South Africa is ~25%, according to the Global Antimicrobial Resistance Surveillance System (GLASS) reports [19–21]. The latest GLASS report (2020) showed that the median rate observed for bloodstream related MRSA infections globally was 12.11% [interquartile range (IQR): 6.4 to 26.4], however, the report does state that the data are not nationally representative and should be interpreted with caution [21].
A previous South African study conducted from 2013 to 2016 in five public tertiary hospitals in two provinces [Gauteng (GAU) and the Western Cape (WC)] showed that the majority of MRSA bacteraemia cases were healthcare-associated (HA) [18]. The most prevalent spa-types identified in the studied period were t037, t045 and t1257 [18].
The aim of this study was to determine if HA-MRSA isolates (with the same spa-type) collected from 2013 to 2017 from two geographically distinct hospitals in South Africa were genetically related based on PFGE. Furthermore, a small subset of MRSA isolates, that were selected based on spa-type, PFGE clustering, time and place of isolation, were characterised with WGS and then compared to PFGE to determine if PFGE is still a reliable method to define outbreaks and/or clonal transmission. This study will shed light on the major MRSA clones in circulation as well as their genetic characteristics.
Materials and methods
Case definitions, study setting and ethical approval
A case of HA-S. aureus bacteraemia was defined as the isolation of S. aureus from a BC after 48 h of admission. Methicillin-resistance was defined as non-susceptibility to oxacillin [minimum inhibitory concentration (MIC) ≥ 4 μg/mL] and polymerase chain reaction (PCR) detection of the mecA gene. Cases originated from two public tertiary academic hospitals; one situated in the WC province and another in the GAU province. A few HA-MRSA isolates in this study originated from a previously published sentinel site surveillance study (January 2013 to January 2016) [18]. Surveillance started in the GAU hospital in 2013 (i.e. five-year period), whereas surveillance in the WC hospital started in 2014 (i.e. four-year period). The study was approved by the Human Research Ethics Committee of the University of the Witwatersrand (Protocol No: M10464) as part of an already established surveillance system (GERMS-SA) for pathogens of public health importance at the National Institute for Communicable Diseases (NICD).
Phenotypic characterisation
Isolates were submitted on Dorset-egg transport media [Diagnostic Media Products (DMP), National Health Laboratory Service (NHLS), SA] from two regional public diagnostic laboratories to the Antimicrobial Resistance Laboratory and Culture Collection (AMRL-CC) in the Centre for Healthcare-associated infections, Antimicrobial Resistance and Mycoses (CHARM), NICD. Isolates were identified with the VITEK 2 system (bioMérieux, France) in 2013. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) (Microflex, Bruker Daltonics, USA) was used for identification from 2014 onwards. Antimicrobial susceptibility testing (AST) was done with the MicroScan Walkaway 96 plus system (Beckman Coulter, USA) and interpreted according to the Clinical Laboratory Standards Institute (CLSI) guidelines (2017) [22].
Total genomic DNA extraction
Total genomic DNA was extracted using a crude boiling method. Colonies were re-suspended in 400 μL tris-ethylenediamine tetra-acetic acid (Tris-EDTA) buffer (10 mM:1mM, pH 8) and boiled at 95°C for 25 minutes, followed by centrifugation. The supernatant was stored at -20°C until used for molecular PCR testing.
PCR detection and typing techniques
The mecA and the S. aureus species-specific nuc genes were detected on the LightCycler 480 instrument II (Roche Life Science, Germany) using previously published primer and probe sequences [23]. S. aureus ATCC 49476 was used as a positive control for the mecA and nuc PCR assay. All MRSA isolates underwent SCCmec typing using the multiplex PCR strategy devised by Milheiriço et al. [24,25]. The following S. aureus strains were used as positive controls for the SCCmec PCR assay as previously published [25]: COL (SCCmec I and ccr class 1), BK2464 (SCCmec II, ccr class 2 and mec class A), ANS46 (SCCmec III and ccr class 3), MW2 (SCCmec type IV and mec class B), WIS (SCCmec V, ccr class 5 and mec class C) and HDE 288 (SCCmec VI and ccr class 4). Spa-typing was done by amplifying the spA locus, followed by PCR purification and Sanger sequencing [26]. Sequence assembly was done in the CLC Main Workbench (QIAGEN, Netherlands) and specific spa-types were assigned with the Ridom StaphType software (Ridom GmbH, Germany) [26]. Unidentified spa-types were submitted to the spa-typing website (http://www.spaserver.ridom.de/), which is curated by SeqNet.org (http://www.SeqNet.org/).
The location (GAU vs. WC); followed by the detected spa-type, the detected SCCmec type, methicillin-resistance and isolate number were the conventional nomenclature used to describe isolates in this study [i.e. (location)-(spa-type)-(SCCmec-type)-(MRSA)-(isolate number)].
Pulsed-field gel electrophoresis
HA-MRSA isolates with the same spa-type occurring in the same hospital, in similar wards (i.e. adult and paediatric wards) were selected for PFGE. PFGE was done as previously described [27,28]. Cluster analysis was done in BioNumerics (GelCompar II Gel Electrophoresis Software, Version 7.6, Applied Math, UK). Pairwise similarity values were calculated with the band-based Dice coefficient with optimisation and a band matching tolerance set at 1.5%. Unweighted pair group matching analysis (UPGMA) was the clustering method applied. Isolates grouping with a similarity value of ≥80% were considered genetically related.
Whole-genome sequencing and analysis
To supplement PFGE, WGS was performed on 21 selected HA-MRSA isolates to confirm the genetic relatedness. The selection was based on PFGE clustering (i.e. showed ≥ 80% sequence similarity), time (specimen collection date) and place (hospital and residing ward) of isolation for MRSA isolates with the same spa-type. The genomic DNA from each isolate was extracted using the QIAamp DNA Mini Kit (Qiagen, Germany) with the inclusion of lysozyme (10 mg/mL) to ensure sufficient lysis. Library preparation was done with the Nextera DNA Flex library prep kit (Illumina, USA) and sequencing was done on the MiSeq platform (Illumina, USA) at a 2x300 bp read length at a 100x coverage.
Raw sequencing reads were analysed using the Jekesa pipeline (v1.0; https://github.com/stanikae/jekesa). Briefly, Trim Galore! (v0.6.2; https://github.com/FelixKrueger/TrimGalore) was used to filter the paired-end reads (Q>30 and length >50 bp). De novo assembly was performed using SPAdes v3.13 [29] and the assembled contigs were polished using Shovill (v1.1.0; https://github.com/tseemann/shovill). The multilocus sequence typing (MLST) profiles were determined using the MLST tool (v2.16.4; https://github.com/tseemann/mlst). Assembly metrics were calculated using QUAST (v5.0.2; http://quast.sourceforge.net/quast). Whole-genome single nucleotide polymorphism (SNP) differences were determined with a reference-free approach using the SKA toolkit [30]. The SNP cut off value of ≤20 was used as previously published [31]. Antibiotic resistance profiles and virulence genes were predicted using ResFinder [32], PointFinder [33], VirulenceFinder [34] and NCBI AMRFinderPlus [35], implemented in the Jekesa pipeline. The Center for Genomic Epidemiology web tools (https://cge.cbs.dtu.dk/services/) were used to determine the spa-types, the SCCmec types and the incompatibility (Inc) groups of the selected MRSA isolates. Pathogenwatch (https://pathogen.watch/) was used to construct the phylogenetic tree [Newick (NWK) file]. The exported NWK file was used in Microreact (https://microreact.org/showcase) to visualise and edit the phylogenetic tree. The assembled genome files were submitted to the National Center for Biotechnology Information GenBank and are available under BioProject number: PRJNA686123.
Results
Case contribution per hospital, the percentage of methicillin-resistance and phenotypic characterisation of the MRSA isolates
A total of 529 S. aureus isolates were submitted from 2013 to 2017 from the GAU hospital, whereas 710 S. aureus isolates were submitted from 2014 to 2017 from the WC hospital. Eighteen percent (n = 95/529) and 33% (n = 234/710) of S. aureus isolates were methicillin-resistant in the GAU- and the WC hospitals, respectively (Table 1). The median age of adult patients with MRSA infection in the GAU- and WC hospital was 44 years (IQR: 33–63) and 43.5 years (IQR: 30–60), respectively. The median age of paediatric patients with MRSA infection in the GAU- and WC hospital was 20 days (IQR: 10–63) and 12 days (IQR: 7–40), respectively. The number of MRSA cases detected in both adult and paediatrics patients in the GAU- and WC hospital was 14 in 2013, 76 in 2014 and 2015, 77 in 2016 and 86 in 2017. The source of bacteraemia (i.e bacteraemia without focus) could not be established for the majority of MRSA cases in the GAU and WC hospitals (n = 238/329). The antimicrobial susceptibility profiles of all MRSA isolates from the GAU- and WC hospital is also shown in Table 1.
Spa-type diversity of MRSA isolates, patient demographics of major spa-types and its associated SCCmec types
Thirty different spa-types were observed in the WC hospital. Spa-type t037 (23.9%, n = 56/234) and t045 (23.5%, n = 55/234) were the predominant spa-types in circulation, followed by t012 (17.5%, n = 41/234), t1257 (12.4%, n = 29/234) and t032 (8.1%, n = 19/234). Twenty spa-types (t015, t0121, t223, t238, t294, t304, t324, t432, t498, t578, t1467, t2409, t2526, t5483, t5691, t6330, t6931, t11775, t11775 and t18226) were singletons. One spa-type could not be determined (even after multiple Sanger sequencing attempts). The unknown spa-type was also identified as a singleton. The remaining spa-types [t1971 (2.1%, n = 5/234); t1476 (1.3%, n = 3/234); t018 (0.9%, n = 2/234); t021 (0.9%, n = 2/234) and t718 (0.9%, n = 2/234)] in the WC hospital occurred at low frequencies.
Sixteen different spa-types were observed in the GAU hospital. The predominant spa-types in circulation in the GAU hospital were t037 (43.2%; n = 41/95), t1257 (23.2%, n = 22/95) and t045 (11.6%, n = 11/95). Ten spa-types were singletons (t008, t186, t355, t463, t718, t913, t1096, t4410, t5691 and t19935). This is the first report of the novel spa-type t19935. Spa-type t012 (6.3%, n = 6/95), t022 (3.2%, n = 3/95) and t064 (2.1%, n = 2/95) occurred at low frequencies. Spa-type t037-MRSA cases in the WC hospital was associated with an adult patient population, whereas t037-MRSA cases in the GAU hospital were associated with a paediatric patient population. The SCCmec type III was associated with t037-MRSA cases, whereas unknown SCCmec elements (typed with the Milheiriço’s assay [24,25]), were associated with most of the t045-MRSA cases for both hospitals (Table 1).
Ward distribution among spa-types and pulsed-field gel electrophoresis
The GAU-t1257-IV-MRSA (n = 22/22) and GAU-t045-I-MRSA (n = 11/11) cases occurred in different wards throughout the hospital. However, the GAU-t037-MRSA cases, with diverse SCCmec types (III/IV) occurred mostly in three paediatric wards [34.2% (n = 14/41) in neonatal ICU (NICU), 24.4% (n = 10/41) paediatric surgery ward (PSW) and 14.6% (n = 6/41) paediatric medical ICU (PMICU)]. Therefore, these 30 GAU-t037-MRSA paediatric cases, with diverse SCCmec types (III/IV) and three additional cases (t037-III-MRSA paediatric patients that moved between wards) were selected for PFGE. The WC-t1257-I-like/II/III/IV-MRSA (n = 29/29), WC-t032-I-like/IV-MRSA (n = 17/17) and WC-t012-II/III-like/IV-MRSA (n = 38/38) cases occurred in different adult and paediatric wards in the hospital, however, 69% (n = 38/55) of the WC-t037-II/III-MRSA cases occurred on the same floor where the adult surgical ICU (SICU) and burn unit were located. Therefore, these 38 WC-t037-II/III-MRSA adult cases were selected for PFGE.
The dendrogram for both WC- and GAU-t037-MRSA cases, with diverse SCCmec types (II/III/IV) is shown in Fig 1. A single isolate (WC-t037-II-MRSA-9025) clustered separately from all cases [similarity value (SV) = 55.8%]. The remaining GAU- and WC-t037- MRSA cases were genetically similar (SV = 84.8%) that branched into three clusters (SV = >90%). The one group (SV = 91.8%) consisted of only WC-t037-III-MRSA cases, whereas the other group (SV = 91.5%) consisted of both WC- and GAU-t037-III-MRSA cases. Numerous WC- and GAU-t037-III-MRSA cases were genetically indistinguishable (SV = 100%).
• = Isolates underwent WGS; NICU = Neonatal ICU; SICU = Surgical ICU; PMICU = Paediatric medical ICU; PSW = Paediatric surgery ward; PW = Paediatric ward; WC = Western Cape; GAU = Gauteng.
Seventy-six percent (n = 41/54) of the WC-t045-MRSA cases occurred in paediatric wards [paediatric haematology ward (PHAE) (40.7%, n = 22/54), neonatology ward (NN) (16.7%, n = 9/54), paediatric ICU (PCIU) (11.1%, n = 6/54), neonatal high care ward (NHC) (3.7%, n = 2/54), paediatric ward (PW) (3.7%, n = 2/54)] and eight paediatric WC-t045-I-MRSA patients moved between these wards. Therefore, 49 WC-t045-I-MRSA paediatric cases were selected for PFGE.
The dendrogram for WC-t045-I-MRSA cases is shown in Fig 2. Cases grouped into two genetically similar clusters (SV = 84.7% and SV = 94.8%) and numerous cases within each cluster were genetically indistinguishable (SV = 100%).
• = Isolates underwent WGS; NN = Neonatology, NHC = Neonatal high care; PHAE = Paediatrics haematology; PICU = Paediatric ICU; PW = Paediatric ward; WC = Western Cape.
Whole-genome sequencing analysis
All the WC-t045-I-MRSA isolates belonged to sequence type (ST) 5 and are part of the clonal complex (CC) 5, which is also known as the pandemic British EMRSA-3 clone (ST5-I-MRSA). All the GAU- and WC-t037-III-MRSA isolates belonged to ST239 and are part of CC8, which is also known as Vienna/Hungarian/Brazilian clone (ST239-III-MRSA). The spa-type and SCCmec type data were in accordance with the WGS results.
The GAU- and WC-t037-III-MRSA harboured the RepA_N (rep20; n = 9/9), Rep1 (rep21; n = 9/9); Rep_trans (rep7a; n = 8/9); RepL (rep10; n = 2/9) and Rep3 (rep5a; n = 1/9) Inc groups. The WC-t045-I-MRSA isolates harboured the Rep1 (rep21; n = 12/12) and RepL (rep10; n = 4/12) Inc groups. All GAU- and WC-t037-III-MRSA isolates as well as the WC-t045-I-MRSA isolates harboured the Rep1 (rep21; n = 21/21).
All MRSA isolates that underwent WGS (n = 21/21) were susceptible to fusidic acid, vancomycin, daptomycin, rifampicin, moxifloxacin and teicoplanin but were resistant to all aminoglycosides (amikacin, gentamicin, tobramycin, kanamycin) (Fig 3). All MRSA isolates (n = 21/21) contained the aacA-aphD (gentamicin, tobramycin and kanamycin) and mecA (methicillin and penicillin) antibiotic resistance genes as well as the haemolysins (hlgA, hlgB and hlgC genes), leukocidins (lukD and lukE genes), serine proteases (splA and splB) and aureolysin (aur gene) virulence genes. All GAU- and WC-t037-III-MRSA isolates contained the following SNPs: (i) ileS-1 (V588F); (ii) grlA (S80F) and (iii) gyrA (S84L). These SNPs gave rise to mupirocin and ciprofloxacin resistance (Fig 3).
The phylogenetic tree and timeline was drawn with Microreact version 92.0.0 (https://microreact.org/project/sbBQtaWKnKdCHZ6L7bbQgJ/a639f664). NICU = Neonatal ICU; PMICU = Paediatric medical ICU; PSW = Paediatric surgery ward; PHAE = Paediatrics haematology; NN = Neonatology; PICU = Paediatric ICU; SICU = Surgical ICU. Reprinted from https://microreact.org/project/sbBQtaWKnKdCHZ6L7bbQgJ/a639f664 under a CC BY licence, with permission from the Centre for Genomic Pathogen Surveillance.
The GAU-t037-III-MRSA-7501 (isolated in 2013—first isolate collected) and GAU-t037-III-MRSA-12725 (isolated in 2017—the last isolate collected) had 211 SNP differences, whereas the WC-t045-I-MRSA-7840 (isolated in 2014—first isolate collected) and WC-t037-III-MRSA-12765 (isolated in 2017—the last isolate collected) had 11296 SNP differences. The SNP differences are provided in S1 and S2 Tables.
The SNP differences (0 to 18) between GAU- and WC-t037-III-MRSA isolates as well as WC-t045-I-MRSA isolates are shown in S3 Table. It is evident that the same GAU-t037-III-MRSA clone is circulating between the PMICU and PSW, the WC-t037-III-MRSA clone is circulating between the burn unit and the SICU ward and the WC-t045-I-MRSA clone is circulating between PHAE, PICU and NN wards.
Discussion
We investigated the genetic relatedness of HA-associated MRSA isolates at two hospitals in South Africa; as the MRSA cases shared the same spa-type and occurred in adult and paediatric wards in the respective hospitals. PFGE and WGS were used to genetically characterise the MRSA isolates.
PFGE showed that t037-III-MRSA and t045-I-MRSA isolates were genetically indistinguishable. PFGE and WGS results were mostly comparable and PFGE can still be used as a reliable method to determine transmission chains and outbreaks in low- to middle-income countries. However, WGS is recommended, as it provides more information on the genetic diversity of the isolates.
WGS showed that all the identified plasmids in this study only carried one rep gene sequence. S. aureus plasmids that carry more than one rep gene sequence has been identified previously [37,38]. There is currently no clear understanding of how virulence- and resistance genes are linked to rep genes and plasmids in S. aureus [37]. The most dominant rep genes detected in this study was rep20 (carried on the RepA_N Inc group), rep21 (carried on the Rep1 Inc group) and rep7a (carried on the Rep_trans Inc group). Rep5, rep7, rep10, rep20 and rep21 was also described in human, animal and food S. aureus isolates of which rep10 and rep21 was the most dominant rep gene sequences [39]. The previously mentioned rep genes except for rep5 were also detected in a study conducted by Strommenger et al. in 174 S. aureus isolates from 33 different countries on five continents from 1957 to 2008 [38].
Two pandemic clones [i.e. Vienna/Hungarian/Brazilian clone (ST239-MRSA-III) and British EMRSA-3 clone (ST5-MRSA-I)] were in circulation in the two studied hospitals and a low frequency of SNP differences (SNPs ≤20) was observed among isolates with the same spa-type occurring in adult and paediatric wards. A definite epidemiological SNP cut-off value to determine strain relatedness in MRSA is not yet established, but low core genome SNP values (≤20) are suggestive of relatedness and therefore a recent transmission event originating from the same source [31,40,41]. A study by Harris et al. [40] estimated that one core SNP mutation occurs roughly every six weeks, which equates to the accumulation of roughly eight to nine SNP differences per year within MRSA’s genome [40]. A study by Ankrum and Hall [41] classified S. aureus strains as the same if ≤71 SNP differences were observed. Another study by Goyal et al. [31] recommended a median cut-off value of 20 SNP differences [31].
The low frequency of SNPs observed among: i) paediatric GAU-t037-III-MRSA isolates, ii) among the surgical and burn adult WC-t037-III-MRSA isolates and iii) among paediatric WC-t045-I-MRSA isolates, are all strongly suggestive that the same strain has been in circulation throughout the study period (i.e. 2013/2014 to 2017). There is some evidence in the literature that t045-MRSA-I and t037-MRSA-III strains have been in circulation for an even longer period, as a study by Moodley et al. [42] reported the circulation of ST5-t045-MRSA-I isolates in the WC and ST239-t037-MRSA-III isolates in GAU, from a set of 320 MRSA isolates originating from various clinical specimens collected from 2005 to 2006 across public and private laboratories in South Africa.
The uninterrupted transmission of the same strain type indicates a lapse in infection prevention and control, which is potentially suggestive of asymptomatic carriage by healthcare workers (HCWs). MRSA can survive on dry surfaces for an extended period and can contaminate the hands of HCW from where it can be transmitted to patients [43,44]. Another potential reason for the uninterrupted transmission is overcrowding of hospital wards and staff shortages. In South Africa, a survey of public medical and surgical unit nurses showed that the nurse-to-patient ratio ranged between 1:8.75 and 1:32 [45], whereas a national audit showed that there are 0.3 trained ICU nurses per ICU/high-care bed [46]. The adoption of routine screening of HCWs as part of occupational health and safety, followed by decolonisation, as well as deep environmental cleaning and hand hygiene compliance, to eradicate MRSA is recommended [43,44,47]. The implementation of such policy measures will disrupt the continuous transmission of the same strain and will lead to a decreased prevalence of MRSA. However, routine screening of HCWs are part of policies in high-income countries and implementation in a resource-poor setting might pose additional challenges, as HCWs colonised with MRSA will need to be placed on paid sick leave until successfully decolonised and might place additional pressure on an already short-staffed unit [47].
The study has some limitations. The study was retrospective and a secondary analysis of a previously reported surveillance programme. The definite source of MRSA could not be established as the genetic relatedness between the cases were detected after the completion of the surveillance period. In addition, only MRSA isolates underwent spa-typing and some transmission events involving MSSA could have been missed. Also, the presence of the Panton–Valentine leukocidin (pvl) gene in the study isolates was not investigated as it was not part of the aim of the study.
Supporting information
S1 Table. SNP differences between MRSA-t037 isolates submitted for WGS.
https://doi.org/10.1371/journal.pone.0253883.s001
(DOCX)
S2 Table. SNP differences between MRSA-t045 isolates submitted for WGS.
https://doi.org/10.1371/journal.pone.0253883.s002
(DOCX)
S3 Table. SNP differences between t037-III and t045-I-MRSA isolates (SNP cut off value ≤20 SNP differences).
https://doi.org/10.1371/journal.pone.0253883.s003
(DOCX)
Acknowledgments
We thank the laboratory staff for submitting isolates, patients for participating in the surveillance, surveillance officers for collecting clinical data, and all other individuals who contributed to the Group for Enteric, Respiratory and Meningeal Surveillance in South Africa (GERMS-SA). Members of the study group are Charlotte Sriruttan, Norma Bosman, and Trusha Nana (Charlotte Maxeke Johannesburg Academic Hospital); Gary Reubenson (Helen Joseph Hospital–Ranmini Kularatne, Rahima Moosa Mother and Child Hospital); Anwar Hoosen, Ruth Lekalakala, and Kathy Lindeque (Steve Biko Pretoria Academic and Tshwane District Hospitals); Vanessa Quan, Susan Meiring, Nevashan Govender, Sonwabo Lindani, Mmakgomo Rakhudu, Penny Crowther and Melony Fortuin-de Smidt (National Institute for Communicable Diseases). The authors would like to thank all staff members of the Antimicrobial Resistance Laboratory and Culture Collection (AMRL-CC), specifically Marshagne Smith, Gloria Molaba, Rosah Kganakga, Rubeina Badat, Naseema Bulbulia, Crystal Viljoen and Cheryl Hamman for phenotypic testing, Boniwe Makwakwa for data capturing and Ruth Mogokotleng for molecular testing. We would also like to thank the staff members of the Sequencing Core Facility (SCF), Senzo Mtshali for performing the library preparation and sequencing and Mushal Allam for reviewing our manuscript.
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