Molecular epidemiology of Methicillin-resistant Staphylococcus aureus in Africa: a systematic review

Methicillin-resistant Staphylococcus aureus (MRSA) infections are a serious global problem, with considerable impact on patients and substantial health care costs. This systematic review provides an overview on the clonal diversity of MRSA, as well as the prevalence of Panton-Valentine leukocidin (PVL)-positive MRSA in Africa. A search on the molecular characterization of MRSA in Africa was conducted by two authors using predefined terms. We screened for articles published in English and French through to October 2014 from five electronic databases. A total of 57 eligible studies were identified. Thirty-four reports from 15 countries provided adequate genotyping data. CC5 is the predominant clonal complex in the healthcare setting in Africa. The hospital-associated MRSA ST239/ST241-III [3A] was identified in nine African countries. This clone was also described with SCCmec type IV [2B] in Algeria and Nigeria, and type V [5C] in Niger. In Africa, the European ST80-IV [2B] clone was limited to Algeria, Egypt and Tunisia. The clonal types ST22-IV [2B], ST36-II [2A], and ST612-IV [2B] were only reported in South Africa. No clear distinctions were observed between MRSA responsible for hospital and community infections. The community clones ST8-IV [2B] and ST88-IV [2B] were reported both in the hospital and community settings in Angola, Cameroon, Gabon, Ghana, Madagascar, Nigeria, and São Tomé and Príncipe. The proportion of PVL-positive MRSA carriage and/or infections ranged from 0.3 to 100% in humans. A number of pandemic clones were identified in Africa. Moreover, some MRSA clones are limited to specific countries or regions. We strongly advocate for more surveillance studies on MRSA in Africa.


Introduction
Methicillin-resistant Staphylococcus aureus (MRSA) is a major public health concern and is responsible for both hospital-and community-associated infections worldwide (De Kraker et al., 2011;CDC, 2013;Falagas et al., 2013;Garza-González and Dowzicky, 2013;Lee et al., 2013;Chen and Huang, 2014). It is estimated that MRSA infections within the health care setting alone affected more than 150,000 patients annually in the European Union, with an additional cost of 380 million Euros (Köck et al., 2010). In the United States of America, 80,461 invasive MRSA infections and 11,285 related deaths occurred in 2011, and an estimated annual burden of between $1.4 billion and 13.8 billion was attributed to community-acquired MRSA (CDC, 2013;Lee et al., 2013). Besides, MRSA has been established as a pathogen for domestic animals and linked with livestockassociated infections (Verkade and Kluytmans, 2013).
Methicillin resistance is usually due to the mecA gene, borne on the staphylococcal cassette chromosome mec (SCCmec) that codes for a 78-kDa penicillin binding protein (PBP2a), with decreased affinity to methicillin and all beta-lactam antibiotics (Chambers, 1997). To date, eleven SCCmec types have been identified (IWG-SCC, 2009). Some cassettes, for example, SCCmec II (53 kb) and SCCmec III (67 kb), are large and possess mobile genetic elements (MGE), such as integrated plasmids (pUB110, pI258, and pT181) and transposons (e.g., Tn554) (Ito et al., 2001), and are frequently associated with hospital-acquired MRSA (Ma et al., 2002;Ito et al., 2004). In contrast, SCCmec IV (21-24 kb) and V (27 kb) are shorter elements, generally susceptible to non-beta-lactam antibiotics, and linked with community MRSA (Chambers and Deleo, 2010). However, the spread of various MRSA clones between the hospital and community settings has made the dichotomous ranking difficult (Deurenberg and Stobberingh, 2008). Recently, a variant mecA gene (named mecC) which is situated on an SCCmec XI element has been described . It has a higher relative affinity for oxacillin as compared with cefoxitin (Kim et al., 2012), and exhibits only 69% sequence similarity at the nucleotide level and 63% amino-acid identity to mecA/PBP2a (Paterson et al., 2014b). Furthermore, based on whole genome sequencing, mutations of the endogenous penicillin-binding proteins (PBP) 1, 2, and 3 in mecA and mecC negative strains have been postulated as a possible alternative mechanism for beta-lactam resistance in MRSA (Ba et al., 2014).
There is great interest in tracking, identifying and understanding the diversity of MRSA in various settings. Currently, the most widely used molecular techniques include Staphylococcus protein A gene typing (spa) and multilocus sequence typing (MLST). Studies (particularly using MLST) have provided evidence that a small set of lineages, clonal complex (CC)5, CC8, CC22, CC30, and CC45, are associated with most of the MRSA infections in hospitals (Stefani et al., 2012). Besides, a number of different geographically distinct lineages, CC1, CC8, CC30, and CC80, have also been associated with community MRSA infections (Chatterjee and Otto, 2013), while CC8 and CC30 have been identified as pandemic lineages both in the hospital and community setting (Chatterjee and Otto, 2013). Furthermore, regional clones have been described in Australia (sequence type [ST] 93) (Coombs et al., 2009), India (ST772) (D'Souza et al., 2010;Shambat et al., 2012), South Korea (ST72) (Kim et al., 2007), Taiwan and China (ST59) (Chen and Huang, 2014).
The distribution of MRSA clones in Africa is not welldescribed. Understanding the molecular epidemiology of MRSA in Africa is important as a recent review indicated that since the year 2000, the prevalence of MRSA appears to be increasing in many African countries and pose a visible threat to the continent (Falagas et al., 2013). Furthermore, there is evidence of the replacement of existing MRSA clones with different and new clonal types in a number of countries (Conceição et al., 2007;Aires-de-Sousa et al., 2008;Albrecht et al., 2011;Espadinha et al., 2013;Lim et al., 2013;Nimmo et al., 2013) but information on this trend is lacking in Africa. The occurrence and changes in clonal identities, and their geographic spread is important to understand the spread and evolution of MRSA.
The Panton-Valentine Leukocidin (PVL) is a two-component pore-forming toxin with cytolytic activity on defined cells of the immune system (neutrophils, macrophages and monocytes) (Löffler et al., 2010;Yoong and Torres, 2013). It is encoded by the lukS-PV and lukF-PV genes (Boakes et al., 2011), and PVL-producing S. aureus exhibit a propensity for causing mainly severe and often recurrent skin and soft tissue infections (Shallcross et al., 2013). In addition, PVL-positive MRSA are associated with community onset-pneumonia (Vandenesch et al., 2003). Although the PVL genes are mainly carried by communityassociated MRSA (CA-MRSA) (Vandenesch et al., 2003), data from West and Central Africa showed that at least 40% of clinical methicillin-susceptible S. aureus (MSSA) isolates in this region are PVL-positive (Breurec et al., 2011a;Schaumburg et al., 2011;Shittu et al., 2011;Egyir et al., 2014a). Therefore, the acquisition of the mecA gene by PVL-positive MSSA and the possible dissemination of PVL-positive CA-MRSA could present a significant challenge in disease management and infection control in resource-limited countries in Africa.
This systematic review examined published literature on the molecular epidemiology of MRSA in Africa. By summarizing currently available data on the continent, our objective was to describe the distribution of MRSA clones, the prevalence of PVLpositive MRSA, and to highlight the need to develop more comprehensive surveillance and reporting systems for multidrugresistant organisms such as MRSA in Africa.

Methods
This systematic review was conducted in accordance with the preferred reporting items for systematic reviews and meta-analyses (PRISMA) guidelines (Moher et al., 2009).

Literature Search Strategy
The relevant English and French articles available in five electronic databases (MEDLINE, EBSCOhost, ISI Web of knowledge, Scopus, and African Journals Online) were retrieved by two authors using predefined search terms (Table S1). The literature search was conducted until 31 October 2014. Figure 1 summarizes the study selection process. All duplicate articles were removed and data on MSSA as well as in-vitro studies were also excluded. The eligibility of published reports in this review was based primarily on polymerase chain reaction (PCR) detection of the mecA gene, and the use of at least one molecular tool for genotyping of MRSA strains (Table 1). In addition, worldwide surveys that covered African countries were also included. An MRSA clone was defined based on the combination of MLST sequence type (ST) and SCCmec typing data as previously reported (Okuma et al., 2002). The nomenclature of the SCCmec types was as proposed by the International Working Group on the Classification of Staphylococcal Cassette Chromosome Elements (IWG-SCC, 2009). SCCmec elements that could not be classified were indicated as non-typeable (SCCmec-NT). In this study, we categorized MRSA into various CCs according to the current eBURST scheme, Version 3 (accessed 30 October 2014) (eBURST, www.mlst.net, V3) 1 .

Data Extraction and Synthesis
The relevant data were extracted from each of the articles as stated in Table 1. Separate articles that analyzed the same S. aureus isolates but answered different questions were considered as a single study.

eBURST Analysis
The relationship between the MRSA STs described in this review and other lineages reported world-wide was analyzed using the eBURST scheme. The allelic profiles were downloaded from the MLST website (http://saureus.mlst.net/) which included the African MRSA STs as well as 223 representative and randomly selected STs (from each CC) based on the differences in their allelic profiles. The minimum spanning tree was constructed by the goeBURST algorithm using the Phyloviz software v1.1 (http:// www.phyloviz.net/). 1 eBURST [Online]. Available online at: http://www.mlst.net version 3.

Literature Search
The systematic search of the five electronic databases yielded 1775 articles (Figure 1). No additional studies were identified from AJOL. After the removal of duplicate studies and assessment of titles and abstracts, 110 full-text articles were screened, of which 57 studies were considered eligible for the qualitative analysis according to our inclusion criteria.

Characteristics of the Studies Included in the Systematic Review
Most of the data analyzed were obtained from single center studies conducted mainly in five countries; Tunisia (n = 13), Nigeria (n = 9), South Africa (n = 7), Algeria (n = 5), and Egypt (n = 3) ( Table 1). Multicenter studies were only reported in two articles (Goering et al., 2008;Breurec et al., 2011b), including a survey which comprised five African countries, Cameroon, Madagascar, Morocco, Niger and Senegal (Breurec et al., 2011b), and an inter-continental multicenter study, which included South Africa (Goering et al., 2008). Only three studies investigated the detection of MRSA in animals ( Table 1).
In most of the reports included in this study, S. aureus was identified by phenotypic and culture characteristics, while molecular identification (16S rRNA, detection of the thermonuclease Frontiers in Microbiology | www.frontiersin.org Frontiers in Microbiology | www.frontiersin.org Frontiers in Microbiology | www.frontiersin.org   An international multicenter study which included only South Africa; *Study was conducted in both animal and human host. Frontiers in Microbiology | www.frontiersin.org and the elongation factor tunuc, tuf -genes) was performed in only 12.3% (7/57). The screening for antibiotic resistance and toxin/virulence genes were carried out in seven and 22 studies, respectively (Table 1). Furthermore, all the eligible studies analyzed MRSA using at least one genotyping technique, and 59.6% (34/57) provided adequate genotyping data on MRSA clones from 15 African countries (Tables 1, 2). Studies included in this systematic review did not investigate on the mecC gene.

Detection of Panton-Valentine Leukocidin (PVL) Genes
The screening for PVL-associated genes (lukF-PV and lukS-PV) was carried out in 44 studies, and the detection of PVL genes was only reported in 32 studies ( Table 1). In animals, PVLpositive MRSA (ST5) was described in nasal samples of pigs from Senegal (Fall et al., 2012). In humans, the proportion of PVLpositive MRSA carriage and/or infections ranged from 0.3 to 100%. Studies from Algeria and Tunisia reported higher PVL prevalence while investigations from South Africa reported the lowest prevalence (Table 3). Overall, PVL-positive MRSA were more frequently reported with skin and soft tissue infections, and community-associated clones (Tables 1, 2). There was no report on the role of PVL in necrotizing pneumonia caused by MRSA in Africa.

Clonal Complex 5
This clonal complex is considered the largest group based on the eBURST scheme (Figure 3). It was subdivided into three main clusters and designated as CC5-ST1, ST5, and ST8.

Clonal Complex 45
ST45-IV [2B], the "Berlin clone, " was detected in an hospital in the KwaZulu-Natal (South Africa) during a multicenter surveillance study (Essa et al., 2009) and ST45-V [5C] was reported in mother-infant pairs in Gabon . An MRSA with a non-typeable SCCmec associated with community-acquired infections has been identified in Tunisia (Ben Jomàa-Jemili et al., 2013). Finally, ST508-V [5C], a slv to ST45, and also associated with community-acquired infections was described in Ghana (Egyir et al., 2014a).

Discussion
MRSA has been reported in Africa, at least since 1978 (Scragg et al., 1978). This systematic review showed that adequate data on the molecular epidemiology of MRSA are limited, with reports from only 15 of the 54 African countries. No spa type was dominant, however, t042 and t044 were the major spa types identified in three North African countries (Table 2). Moreover, we did not observe a clear distinction between hospital-and community-associated MRSA clones in Africa which is in agreement with other investigations worldwide (Fossum Moen et al., 2013;Pasquale et al., 2013;Sherwood et al., 2013;Tavares et al., 2013). In this systematic review, the use of the current eBURST scheme grouped several African MRSA CCs (CC1, CC5, CC8, and CC7) into a single cluster, (CC5). This raises some concern on a suitable method for discrimination and grouping of S. aureus isolates. To overcome the above mentioned issue, whole genome sequencing approach might be the alternative (Dabul and Camargo, 2014).
Although a combination of factors could be responsible for the dissemination of clones between continents, increased movement of human population within or between countries might be one of the potential factors (Rogers et al., 2011). International travel could play a significant role in the transmission of MRSA, particularly the replacement of existing MRSA with fitter and more transmissible clones (Zhou et al., 2014). We observed that the predominant hospital-associated epidemic clones, EMRSA-15 [ST22-IV [2B]) and ), in the United Kingdom (UK) (Johnson et al., 2005) were reported only in South Africa (Goering et al., 2008;Moodley et al., 2010;Jansen van Rensburg et al., 2011;Oosthuysen et al., 2014). Moreover, ST80-IV [2B] (the European clone) has consistently been recognized as the predominant PVL-positive MRSA clone in North Africa (Ramdani-bouguessa et al., 2006;Bekkhoucha et al., 2009;Ben Nejma et al., 2009Enany et al., 2010;Antri et al., 2011;Ben Slama et al., 2011;Ben Jomàa-Jemili et al., 2013;Djoudi et al., 2013). A recent report based on whole genome analysis provided strong evidence that the European ST80-IV [2B] was derived from a PVL-positive MSSA ancestor in sub-Saharan Africa that acquired the SCCmec IV element, and clonal spread was enhanced by increased transnational movement (Stegger et al, 2014). However, the factors responsible for the limited spread of the ST80-IV [2B] only in North Africa observed so far are unclear.
The SCCmec IV (and its subtypes) and SCCmec V were identified in several MRSA clones, and ST5 and ST8 clearly showed more diversity in terms of SCCmec types compared with other STs in Africa. The success of these SCCmec types (IV and V) could be due to their small sizes and low fitness costs (Enright et al., 2002;Okuma et al., 2002;Monecke et al., 2011). It is also noteworthy that the SCCmec types IVa and IVc were identified in genetically unrelated clones, e.g., ST5-IVa [2B] (CC5) in Algeria (Ramdani-bouguessa et al., 2006;Bekkhoucha et al., 2009), São Tomé and Príncipe (Conceição et al., 2013),  in Tunisia (CC5) (Kechrid et al., 2011), and ST37-IVa [2B] (CC30) in Algeria (Ramdani-bouguessa et al., 2006). This might suggest horizontal gene transfer or independent acquisition (Mašlaòová et al., 2013). Another interesting observation was the detection of the SCCmec type IVa and V in the hospitalassociated ST239/ST241-III [3A] in Algeria (Ramdani-bouguessa et al., 2006), Nigeria (Ghebremedhin et al., 2009), and Niger (Breurec et al., 2011b). Since ancient MSSA strains for this ST have not been reported (Enright et al., 2002), our observation suggests that acquisition of these SCCmec types by MSSA is less likely, and points to the possible replacement of SCCmec type III with IV and V on the ST239/241 genome .
Data on the epidemiology of MRSA in animals are limited in Africa (EL Seedy et al., 2012;Fall et al., 2012;Gharsa et al., 2012). Moreover, the genetic relatedness between human and animal MRSA has not been investigated ( Table 2). It should be noted, however, that human-associated ST5-IV [2B], ST88-IV [2B], and ST153-SCCmec-NT have been reported from animals in Tunisia (Gharsa et al., 2012) and Senegal (Fall et al., 2012). Recently, human-associated S. aureus lineages were described in captive Chimpanzees in Gabon, Madagascar, Uganda and Zambia (Schaumburg et al., , 2013Nagel et al., 2013). Notably, a likely case of S. aureus transmission from a veterinarian to a chimpanzee from the same sanctuary was demonstrated . Zoonotic transmission may constitute a major concern in Africa, where there is often substantial exposure to domesticated animals (Fall et al., 2012;Gharsa et al., 2012). Furthermore, animal-adapted clones might undergo further host-adaptive evolutionary changes, which could result in an epidemic spread of new and more virulent strains in the human population (Spoor et al., 2013). Other risk factors for animal to human MRSA transmission, which include contaminated environment (Verkade and Kluytmans, 2013) and meat products (Hamid and Youssef, 2013), have not been investigated in Africa. Livestock-associated MRSA are widespread in Europe, but the transmission of these strains to humans is either rare or limited to people with direct contact with MRSA infected/carrier animals (Verkade and Kluytmans, 2013). Using whole genome sequencing, evidence of zoonotic transmission of MRSA harboring mecC was reported in Denmark (Harrison et al., 2013). The mecC-positive MRSA, initially known as a livestock MRSA belonging to the CC130, is recognized in both animals and humans in Europe (Paterson et al., 2014a). In addition, this clone has been implicated in severe infections in humans (Paterson et al., 2014b), resulting in one death (García-Garrote et al., 2014). The clinical importance of mecC-positive MRSA is not yet clear in Africa as data is unavailable. Therefore, we suggest that surveillance for MRSA should include detection of the mecC gene where mecA is not detected in resistant isolates.
This systematic review did not seek to provide comprehensive information on the burden of PVL-positive MRSA in Africa. However, it provided some interesting observations on their epidemiology in Africa such as the identification of PVL-positive isolates assigned with CC7 (originally classified with CC152) in Ghana (Egyir et al., 2014a), CC88-IV [2B] in Cameroon (Breurec et al., 2011b) and Nigeria (Ghebremedhin et al., 2009), ST612-IV [2B] in South Africa (Oosthuysen et al., 2014), and CC152 in Nigeria . Until now, CC152 was only described in the Balkan region (Francois et al., 2008). The mode of acquisition of the mecA gene by ST152 is still unknown, but it might be explained by either its introduction through international travel or the acquisition of the methicillin resistance gene by PVL-positive MSSA, which is prevalent in West and Central Africa (Ruimy et al., 2008;Okon et al., 2009;Breurec et al., 2011a;Schaumburg et al., 2011;Shittu et al., 2011Shittu et al., , 2012Egyir et al., 2014a). These observations highlight the need for further surveillance data (including information on community-acquired necrotizing pneumonia) to understand the epidemiology of PVL-associated S. aureus in both hospital and community settings on the African continent.

Conclusion
A number of pandemic MRSA clones were identified in Africa. In contrast, some MRSA clones are limited to specific countries or regions. Although the eBURST snapshot provided a description of the relationship between the MRSA clones reported in Africa and other lineages submitted into the MLST database from other continents, the objective of this review was not to understand the origin of MRSA clones in Africa, as this will require in depth analysis like whole genome sequencing. However, it did show that CC5 is the largest group and predominant in Africa. Nevertheless, the limited data available on MRSA in Africa draw attention to the need for increased surveillance of MRSA and molecular epidemiological studies. We strongly recommend improved co-operation between clinicians and microbiologists in Africa. This synergy could provide an understanding on the local epidemiology of MRSA. In addition, we strongly advocate the establishment of effective diagnostic microbiology facilities that will incorporate high-throughput technologies for monitoring the clonal expansion and dissemination of MRSA. In the meantime, increased networking through collaboration with S. aureus reference centers could provide support for genotyping services to African countries with limited resources. Finally, population-based surveillances for MRSA are needed to evaluate the situation of community associated MRSA as well as studies on MRSA from animal hosts. To understand the origin of the newly emerged clones, MSSA genotyping needs to be incorporated with MRSA surveillance studies.

Author Contributions
MK, AS, and SMA initiated the project. SMA extracted the data and reviewed the articles with MK. SMA, AS, MN, and MK wrote the manuscript. All the authors reviewed the final version of the manuscript prior to submission for publication

Financial Support
This systematic review was supported by the Organization for Women in Science for the Developing World; Clinical Infectious Diseases Research Initiative, University of Cape Town, South Africa; Bill and Melinda Gates Foundation Global Health Grant (OPP107641), United States of America; Deutscher Akademischer Austausch Dienst, Germany; Carnegie Corporation of New York, United States of America. Any opinions, findings and conclusions, or recommendations expressed in this review are those of the authors, and therefore do not represent the official position of the funders.