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Molecular Epidemiology and Antimicrobial Susceptibility of Clinical Staphylococcus aureus from Healthcare Institutions in Ghana

Molecular Epidemiology and Antimicrobial Susceptibility of Clinical Staphylococcus aureus from... The objective of this study was to determine the antimicrobial susceptibility patterns and clonal diversity of clinical Staphylococcus aureus isolates from Ghana. A total of 308 S. aureus isolates from six healthcare institutions located across Northern, Central and Southern Ghana were characterized by antibiotyping, spa typing and PCR detection of Panton Valentine leukocin (PVL) genes. Methicillin-resistant S. aureus (MRSA) were confirmed by PCR detection of mecA gene and further characterized by SCCmec and multi-locus sequence typing (MLST). The prevalence of antimicrobial resistance was below 5% for all agents tested except for penicillin (97%), tetracycline (42%) and erythromycin (6%). Ninety-one spa types were found, with t355 (ST152, 19%), t084 (ST15, 12%) and t314 (ST121, 6%) being the most frequent types. Based on established associations between spa and MLST types, isolates were assigned to 16 clonal complexes (CCs): CC152 (n = 78), CC15 (n = 57), CC121 (n = 39), CC8 (n = 36), CC5 (n = 33), CC1 (n = 29), CC45 (n = 9), CC88 (n = 8), CC30 (n = 4), CC9 (n = 3), CC25 (n = 2), CC97 (n = 2) CC20 (n = 2), CC707 (n = 2), CC7 (n = 3) and CC522 (n = 1). Most isolates (60%) were PVL-positive, especially those belonging to ST152, ST121, ST5, ST15, ST1, ST8, and ST88. Nine (3%) isolates were MRSA belonging to seven distinct clones: ST88-IV (n = 2), ST250-I (n = 2), ST8-IV (n = 1), ST72-V (n = 1), ST789-IV (n = 1), ST2021-V (n = 1), and ST239-III (n = 1). The study confirmed a high frequency of PVL-positive S. aureus in Africa, low prevalence of antimicrobial resistance and high diversity of MRSA lineages in Ghana compared to developed countries and other African countries. The detection of known pandemic MRSA clones in the absence of routine MRSA identification in most Ghanaian clinical microbiology laboratories calls for capacity building to strengthen surveillance and prevent spread of these clones. Citation: Egyir B, Guardabassi L, Sørum M, Nielsen SS, Kolekang A, et al. (2014) Molecular Epidemiology and Antimicrobial Susceptibility of Clinical Staphylococcus aureus from Healthcare Institutions in Ghana. PLoS ONE 9(2): e89716. doi:10.1371/journal.pone.0089716 Editor: J. Ross Fitzgerald, University of Edinburgh, United Kingdom Received October 8, 2013; Accepted January 23, 2014; Published February 25, 2014 Copyright:  2014 Egyir 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. Funding: This study was supported by Antibiotic Drug use, Monitoring and Evaluation of Resistance (ADMER) project funded by the Danish International Development Agency (DANIDA). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interest exist. * E-mail: begyir@sund.ku.dk Africa as recently suggested [5,6]. PVL is associated with SSTI Introduction and severe necrotising pneumonia and has been shown to be a Methicillin-resistant Staphylococcus aureus (MRSA) is a major characteristic feature of community acquired (CA) -MRSA clones concern in clinical medicine due to the importance of b-lactams in disseminated in Europe and Middle East (ST-80), Australia and the therapy of staphylococcal infections and the additional South America (ST30-IV), and United States (ST8-IV, also known morbidity and mortality for MRSA patients compared to patients as USA300) [2,7,8]. infected with methicillin-susceptible S. aureus (MSSA) [1]. Despite The objective of this study was to investigate the antimicrobial the importance of MRSA, MSSA are among the most common susceptibility and clonal diversity of clinical S. aureus isolates from causative agents of bacteraemia and skin and soft tissue infections Ghana. Antimicrobial resistance in S. aureus has previously been (SSTI) [2]. Epidemiological surveillance of MRSA and MSSA is of reported from Ghana with findings of a low MRSA prevalence in importance for the development and implementation of infection nasal swabs from patients and health care workers at the Korle-bu control programmes. Data on S. aureus epidemiology in African Hospital, Accra [9] however, treatment in Ghana is mainly countries are limited and a common trait for MSSA strains from empirical due to a relative lack of appropriate laboratory facilities various African countries seems to be the carriage of the PVL [10] and therefore only few susceptibility data exists and so far no genes: lukS/F-pv at much higher frequencies (.55%) than in the study has investigated the clonal structure of S. aureus in clinical rest of the world (,10%) [2–6]. The high frequency of PVL samples. The study was part of a cooperation program on among human MSSA strains is of special interest since the most Antibiotic Drug use, Monitoring and Evaluation of Resistance successful community associated (CA) MRSA clones share this (ADMER) in Ghana under the Danish Ministry of Foreign Affairs. genetic marker, and could have MSSA ancestors associated with PLOS ONE | www.plosone.org 1 February 2014 | Volume 9 | Issue 2 | e89716 Epidemiology of Staphylococcus aureus in Ghana This program was conceived to strengthen clinical microbiology 15 mg erythromycin, 10 mg norfloxacin, 10 mg gentamicin, 10 mg linezolid, 5 mg rifampicin, 1. 25 mg +23.75 mg trimethoprim- and surveillance of antibiotic resistance, and ultimately to improve awareness of antimicrobial use in Ghana. sulfamethoxazole, and 10 mg fusidic acid (Rosco NeoSenstabs, Taastrup, Denmark). Inducible clindamycin resistance was detected by placing clindamycin and erythromycin 12–20 mm Materials and Methods apart (D-test). Brain Heart Infusion agar supplemented with Ethics Statement teicoplanin (5 mg/L) (Becton Dickinson, Denmark) was used to Ethical clearance was obtained from the University of Ghana screen MRSA isolates for glycopeptides resistance by a spot test; if Medical School Ethical and Protocol Review Board (reference no. 10 or more colonies were detected on these plates, E-tests MS-EI/M.9 - P.3.212010-11). (bioMe´rieux, Marcy I’Etoile, France) were used to determine the minimum inhibitory concentration of vancomycin and teicoplanin [11]. Multidrug resistance (MDR) was defined as resistance to at Bacterial Isolates least three distinct antimicrobial classes or being MRSA [12]. Staphylococcal isolates from clinical specimens were obtained in a prospective cross-sectional-like study between October 2010– June 2012 from six healthcare institution situated at Northern Molecular Typing (Tamale Teaching Hospital), Central (Sunyani Government Molecular characterization of the isolates was done at Statens Hospital) and Southern Ghana (Korle bu Teaching Hospital, Serum Institut (SSI), Denmark. A multiplex PCR was used for Thirty-seven Military Hospital, Ridge Hospital and Legon detection of spa, lukS/F-pv and mecA [13]. spa typing was performed Hospital) (Figure 1). The majority of the isolates (70%) were as described by Harmsen et al. [14]. Using BioNumerics v.6.5 obtained from Korle bu Teaching Hospital, which serves a (Applied Maths, Sint-Martens-Latem, Belgium) with the Ridom population of over 3 million and acts as a major referral health spa server (http://spa.server.ridom.de) plug-in, spa sequences were facility for an estimated population of 24 million people across automatically assigned to spa types and clonal complexes (CCs) Ghana. Presumptive staphylococci identified by colony morphol- based on spa repeats. Multi-Locus Sequence Typing (MLST) [15] ogy at the hospital clinical microbiology laboratories were was done on all MRSA and MSSA isolates whose CC could not be collected and sent to Noguchi Memorial Institute for Medical assigned by the Ridom spa server. Minimum Spanning Tree Research, where they were identified as S. aureus by Gram staining, (MST) based on spa- types was made using BioNumerics V6.5 catalase, tube coagulase and slidex staphplus test (bioMe´rieux, (Applied Maths, Sint-Martens-Latem Belgium). Staphylococcal Marcy l’Etoile, France). Available patient demographic charac- cassette chromosome mec (SCCmec) typing was performed by teristics such as age and sex were retrieved from laboratory multiplex PCR as described previously [16]. records. Statistical Analysis Antimicrobial Susceptibility Testing Distributions of the various genotypes determined in the study Susceptibility testing was carried out by disc diffusion technique (PVL-positivity, spa type, ST and CC) were associated to region, following the European Committee on Antimicrobial Susceptibil- hospital, sex and infection type to determine if specific patterns ity Testing (EUCAST) guidelines (www.eucast.org) using 1U existed. Only genotypes with more than 10 observations were penicillin, 30 mg tetracycline, 30 mg cefoxitin, 2 mg clindamycin, included in statistical analysis. MRSA isolates were not evaluated Figure 1. Origins of the 308 clinical Staphylococcus aureus collected from six hospitals in Ghana, 2010–2012. {Referral Hospitals. 1 2 `Secondary Hospitals. 1Primary Hospitals. SSTI: Skin and Soft Tissue Infections. Other: (Urinary Tract Infection (n = 9); Unknown Infections (n = 14)). doi:10.1371/journal.pone.0089716.g001 PLOS ONE | www.plosone.org 2 February 2014 | Volume 9 | Issue 2 | e89716 Epidemiology of Staphylococcus aureus in Ghana due to the low prevalence of their genotypes. Associations were spatial associations were observed in the distribution of spa- determined using the x test, except for PVL-positivity, which was types, STs and CCs with regard to infection type, sex, region analysed by logistic regression. A significant association was and hospital of origin, Some spatial variations were observed in deemed at p-values ,0.05. the distribution of spa types (e.g. t355 occurred in 34% and 13% isolates from Sunyani Government Hospital and Korle bu Teaching Hospital respectively) but such differences could not Results be proven to be significant. Isolates from Ridge Hospital (n = 1), Of the 903 presumed staphylococci collected from the six Thirty-seven Military Hospital (n = 6) and Tamale Teaching hospitals, 308 (34%) were identified as S. aureus and 595 (66%) as Hospital (n = 12) were excluded from the statistical analysis due coagulase negative staphylococci. S. aureus isolates originated from to low numbers. SSTI (n = 173), bacteraemia (n = 112), and other (urinary tract PVL was detected in 60% (n = 184) of the isolates, mainly infection, n = 9; unknown infections, n = 14) infections (n = 23). S. isolates from SSTIs (57%) and belonging to ST152 (38.5%), aureus was isolated from 143 females and 109 males. Sex origin of ST121 (21%), ST5 (13.5%), ST15 (11%), ST1 (7%), ST8 (3.8%) 56 isolates could not be traced from laboratory records. With and ST88 (2.7%). Genotypes and clinical origins of PVL positive regard to hospital origin, 12 isolates were from Tamale Teaching S. aureus are shown in Table 2. The patterns of PVL varied by Hospital (TTH, Northern Ghana), 53 from Sunyani Government region, with Central regions having a higher chance 2.2 (95% CI: Hospital (SGH, Central Ghana) and 243 from the four hospitals in 1.1–4.2) of seeing PVL-positive S. aureus (p = 0.02) than the Southern Ghana. Details of hospital location (stratified into Southern region. regions of study) and proportions of isolates from clinical infections are shown in Figure 1. None of the clinical laboratories used Discussion methods for MRSA detection and typing, and several pitfalls were recognized in routine microbiological procedures (e.g. poor This study fills an important gap in the knowledge of the identification to species/genus level, and low compliance with epidemiology of S. aureus in Ghana. As such, the study international standards for susceptibility testing). contributes to the current knowledge of the diversity and The highest prevalence of resistance was for penicillin (97%), population structure of this important bacterial pathogen at the followed by tetracycline (42%) and erythromycin (6%). Lower global level. Ghana and several other African countries have so percentages of resistance were observed for clindamycin (5%), far been black spots on the map due to lack of established norfloxacin (4%), trimethoprim-sulphamethoxazole (4%), genta- national surveillance programmes and adequate clinical micro- micin (3%), cefoxitin (3%) and fusidic acid (2%). Inducible biology infrastructure [10,17]. Our results show that the most clindamycin resistance was detected among seven (2%) isolates. common spa types among MSSA isolates are t355 (ST152) and Twenty-nine isolates (9%) were MDR, of which 9 (3%) were t084 (ST15). The spa types were previously found to be confirmed mecA positive MRSA. Details of MDR isolates have predominant among S. aureus isolates from asymptomatic nasal been shown in Table 1. MRSA isolates were susceptible to carriers at Korle bu, the largest Teaching Hospital in Ghana vancomycin and teicoplanin. Most of the MSSA isolates (88%, [9], suggesting that they are well established in the human 264/299) were resistant to penicillin (n = 154), penicillin and population of this country. In another African study, t084 tetracycline (n = 99) and penicillin and trimethoprim-sulphameth- (ST15) was also reported as one of the most frequent spa types oxazole (n = 11). All isolates were susceptible to linezolid and among S. aureus isolated from seven tertiary hospitals located in rifampicin while three isolates (1%) were susceptible to all five major African towns [3]. PVL-positive ST152 (t355) is also antimicrobial drugs tested. widely distributed in African countries [4,5] and its frequent High genetic diversity was observed by spa typing, as indicated recovery from SSTI is consistent with studies in other countries by the recovery of 91 spa types among all isolates tested. The most [4,18]. Other PVL-positive MSSA lineages found in this study common spa types were t355 (19%), t084 (12%), t314 (6%) and such as ST121, ST30, ST15 and ST5 have also been reported t311 (5%). Fifty-six spa types were singletons and eight new spa elsewhere in Africa [19]. The observed high prevalence (60%) types were detected: t10809 (ST88), t10810 (ST88), t10811 (ST8), of PVL appears to be a distinguishing genetic trait of African t10828 (ST152), t10833 (ST152), t10836 (ST1), t10837 (ST1) and MSSA [3,4,6] compared to USA, Asia and Europe, where this t10838 (ST30). A minimum spanning tree, including spa types and virulence factor is uncommon in MSSA [2,19,20]. This finding clonal complexes of the 308 isolates is shown in Figure 2. was correlated to the high frequency of PVL-positive ST152, which is a likely ancestor of the CA-MRSA ST152-V clone Based on spa typing, isolates (n = 308) were assigned to 16 MLST clonal complexes: CC152 (n = 78), CC15 (n = 57), CC121 circulating in certain European regions, especially the Balkan (n = 39), CC8 (n = 36), CC5 (n = 33), CC1 (n = 29), CC45 (n = 9), area [21,22]. CC88 (n = 8), CC30 (n = 4), CC9 (n = 3), CC7 (n = 3), CC25 The nine MRSA isolates belonged to seven unrelated spa types (n = 2), CC97 (n = 2) CC20 (n = 2), CC707 (n = 2), and CC522 and STs harbouring four different SCCmec types (Table 1), (n = 1). MRSA isolates belonged to ST88-IV (n = 2), ST8-IV indicating high clonal diversity. Some of the MRSA lineages (n = 1), ST789-IV (n = 1), ST72-V (n = 1) ST2021-V (n = 1), identified in this study are widely distributed worldwide: ST239-III ST250-I (n = 2), and ST239-III (n = 1). Two of them were PVL- is a pandemic clone prevalent in Europe, Asia and South Africa positive and belonged to t121 (ST8) and t547 (ST789) (Table 1). [23–25] and ST789-IV is a single locus variant of the ST7 clone The most common MSSA lineages were ST152 (27%) and ST15 frequently reported in Asia [25]. ST88-IV, ST8-IV and ST72-V (18%). have been previously reported among inpatients and staff at Five spa types (t084, t127, t311, t314 and t355), six STs (ST1, Korle-bu Hospital in Ghana [9] and in communities and hospitals ST5, ST8, ST15, ST121 and ST152) and six CCs (CC1, CC5, in other African countries [26,27]. MRSA ST88 has been reported CC8, CC15, CC121 and CC152) were included in the sporadically in some European countries like Portugal [28] and statistical analysis. Surprisingly, spa type t311 occurred less Sweden [29]. ST8-IV MRSA (spa type t121, PVL+) found in this frequently among females (0.3%) than among males (2%) and study is related to the epidemic MRSA ST8-IV (USA300) clone in those of unknown sex (2.3%) (p = 0.0016). No other clinical or the USA [2]. Other African studies have reported this ST8-IV PLOS ONE | www.plosone.org 3 February 2014 | Volume 9 | Issue 2 | e89716 Epidemiology of Staphylococcus aureus in Ghana Table 1. Origins and characteristics of 29 multi-drug resistant (MDR) Staphylococcus aureus isolated from healthcare institutions in Ghana, 2010–2012. a b c ID Hospital Infection CC ST spa type SCCmec PVL Antibiotype MRSA 5016 KB SSTI CC1 ST72 t537 V 2 Fox, Pen, Tet 744 KB Blood CC8 ST2021 t024 V 2 Fox, Pen, Tet 2244 KB Blood CC8 ST239 t037 III 2 Fox, Pen,Tet, Fuc, Gen, Cli, Ery, 3464 KB Blood CC8 ST8 t121 IV + Fox, Pen, Nor, Cli, Ery 2207 KB SSTI CC8 ST250 t928 I 2 Fox, Pen, Tet, Gen, Nor, Cli, Ery 2224 KB SSTI CC8 ST250 t928 I 2 Fox, Pen, Tet, Gen, Nor, Cli, Ery 44 SGH Unknown CC88 ST88 t186 IV 2 Fox, Pen, Tet AU81 SGH SSTI CC88 ST88 t186 IV 2 Fox, Pen, Tet 11087 KB UTI CC152 ST789 t547 IV + Fox, Pen, Tet, Nor MSSA 2639 KB Blood CC1 ST1 t7835 NA + Pen, Tet, Cli, Ery AU93 SGH SSTI CC1 ST1 t559 NA + Pen, Tet, Fuc A6 KB Unknown CC5 ST5 t311 NA + Pen, Tet, Fuc 5095 KB SSTI CC5 ST5 t071 NA + Pen, Tet, Gen, TMS, Fuc, Nor, Cli, Ery T2845 TTH SSTI CC8 ST8 t451 NA + Pen, Tet, TMS 1455 KB SSTI CC9 ST9 t2700 NA 2 Pen, Gen, Cli, Ery 1050 KB Blood CC15 ST15 t084 NA + Pen, Tet, Gen 2320 KB Blood CC45 ST508 t635 NA 2 Pen, Cli, Ery 1548 KB SSTI CC88 ST88 t10809 NA + Pen, Tet, Nor, Cli, Ery 5270 KB SSTI CC88 ST88 t10810 NA + Pen, Tet, Nor, Cli, Ery NAB KB SSTI CC121 ST121 t213 NA _ Pen, Tet, Fuc 3209 KB Blood CC121 ST121 t091 NA _ Pen, Tet, Nor, Gen, Cli, Ery 2437 KB Blood CC121 ST121 t091 NA _ Pen, Tet, Nor 5293 KB Blood CC121 ST121 t314 NA + Pen, Tet, Nor 5775 KB Blood CC121 ST121 t314 NA + Pen, Tet, Nor 3984 KB SSTI CC152 ST152 t1299 NA + Pen, Tet, Cli, Ery 1544 KB SSTI CC152 ST152 t355 NA + Pen, Tet, Cli, Ery 4836 KB SSTI CC152 ST152 t355 NA + Pen, Tet, Cli, Ery 112242 MH SSTI CC152 ST152 t355 NA + Pen, Tet, Ery A71 SGH Blood CC152 ST152 t355 NA + Pen, Tet, Fuc ST: Sequence Type; CC: Clonal Complex; SCC: Staphylococcal Cassette Chromosome; PVL: Panton- Valentine leukocidin. KB: Korle bu Teaching Hospital; SGH: Sunyani Government Hospital; TTH: Tamale Teaching Hospital; MH: Military Hospital. b c SSTI: Skin and Soft Tissue Infection; UTI: Urinary Tract Infection; TMS: trimethoprim-sulphamethoxazole. Pen: penicillin; Fox: cefoxitin; Tet: tetracycline; Nor: norfloxacin; Gen: gentamicin; Fuc: Fucidic acid; Cli: clindamycin; Ery: erythromycin. doi:10.1371/journal.pone.0089716.t001 MRSA (spa type t121, PVL+) strain in communities and hospitals The prevalence of antimicrobial resistance in clinical S. aureus [26,30]. ST250-I, also referred to as the ‘‘Archaic clone’’, differs isolates from Ghana was generally low. Other African studies have from ST8 by a point mutation in the yqiL gene and is related to reported similar levels of resistance to penicillin (86%–93%) and ST247-I (Iberian clone), a major clone isolated in European tetracycline (28%–48%) but higher levels of resistance to hospitals [7,31]. ST72 has been reported as a major MRSA clone sulphonamides (22%–68%) compared to this study [3,4,9]. from communities in Australia [32] and as MSSA in Nigeria and Comparatively, the prevalence of MRSA (3%) was lower than Gabon [6,27]. The least known MRSA lineage found in this study those reported in other African countries such as Nigeria (20%) was ST2021-V, which to the best of our knowledge has previously [27], Algeria (45%) [33] and in a multicenter study (15%) been reported in a single isolate from Nigeria (www.mlst.net; involving five major African towns [26]. The low MRSA th accessed on: 4 April 2013). Although ST5, ST30 and ST80 frequency reported in this study could be attributed to the low MRSA have been described in several African and other countries consumption of antimicrobial agents such as fluoroquinolones and around the world [26,27], none of these clones were detected third generation cephalosporins in Ghana, because they are among clinical MRSA isolates in Ghana. PVL-positive ST5 and expensive and are usually prescribed for acute infections [10]. ST30 were however detected among MSSA isolates (Table 2), Usage of the afore-mentioned antimicrobial agents has been indicating that these two S. aureus lineages are widespread in shown to correlate with an increase in MRSA prevalence [34–36]. African countries, even though acquisition of methicillin resistance The observed MRSA prevalence among clinical isolates in Ghana seems to be confined to some countries. is similar to those reported in European countries with low MRSA PLOS ONE | www.plosone.org 4 February 2014 | Volume 9 | Issue 2 | e89716 Epidemiology of Staphylococcus aureus in Ghana Figure 2. Minimum spanning tree of 308 clinical Staphylococcus aureus isolates from healthcare institutions in Ghana. Nodes indicate spa types and their size shows the relative number of isolates for each spa type. Numbers of frequent (three or more) spa types have been shown. Every colour represents a distinct clonal complex. doi:10.1371/journal.pone.0089716.g002 Table 2. Clonal complex (CC), multi-locus sequence type (ST), spa type and clinical origin of 184 Staphylooccus aureus harbouring Panton-Valentine leukocidin (PVL) genes isolated in Ghana, 2010–2012. STs and CCs were inferred from spa types. Clinical origin, N (%) CC ST spa type (N) Bacteraemia SSTI Other* Total N=65 N = 104 N=15 N = 184 CC1 ST1 t127 (3), t1931 (3), t693 (1), t559 (1), t10836 (1), 6 (9.2) 7 (6.7) 0 (0.0) 13 (7.0) t114 (1), t922 (1), t934 (1), t7835 (1) CC5 ST5 t071 (9) t311 (9), t002 (5), t105 (1) 12 (18.5) 11 (10.6) 1 (6.7) 24 (13.0) CC8 ST8 t1476 (3), t024 (1), t451 (1), t064 (1), ut121(1) 2 (3.0) 5 (4.8) 0 (0.0) 7 (3.8) CC15 ST15 t084 (19), t5534 (1), t774 (1) 7 (10.8) 13 (12.5) 1 (6.7) 21 (11.4) CC25 ST25 t401 (1) 1 (1.5) 0 (0.0) 0 (0.0) 1 (0.5) CC30 ST30 t10838 (1),t363 (1) 0 (0.0) 2 (1.9) 0 (0.0) 2 (1.1) CC88 ST88 t2393 (2) t3202 (1), t10809 (1), t10810 (1) 1 (1.5) 4 (3.8) 0 (0.0) 5 (2.7) CC121 ST121 t314 (15), t2304 (9), t159 (5), t645 (5), t1077(1), t7002(1) 14 (21.5) 21 (20.2) 1 (6.7) 36 (19.6) CC152 ST152 t355 (57), t4690 (3), t1096 (2), t1299 (2), Singletons (11) 22 (34.0) 41 (39.4) 12 (80.0) 75 (40.8) SSTI: Skin and Soft Tissue Infection;*Other: UTI: Urinary Tract Infection (n = 5: spa types t355 (3), t547(1) and t5534 (1); Unknown (n = 10; spa types: t311 (1), t645 (1), t4690 (1), t355 (7) Other spa types associated with CC152: t454, t458, t5268, ut547, t1123, t1172, t5047, t7011, t8821, t10828, and t10833uMRSA. doi:10.1371/journal.pone.0089716.t002 PLOS ONE | www.plosone.org 5 February 2014 | Volume 9 | Issue 2 | e89716 Epidemiology of Staphylococcus aureus in Ghana prevalence, such as the Scandinavian countries and The Nether- and poor performance standards in most clinical microbiology lands [37]. laboratories in Ghana, highlighting the need for infrastructures Some apparent geographical variations in clonal distribution to support national antimicrobial policies and surveillance were observed, but the low number of isolates obtained from the capacity. Northern region made comparisons between hospitals or regions meaningless. The clinical information on the 308 S. aureus included Acknowledgments in the study varied in quality due to incompleteness of the patient The directors of hospitals are thanked for their support to the study. The records collected from the various hospital clinical laboratories authors are grateful to Dr. Jesper Larsen, Mr Michael Olu-Taiwoo, Mr involved in the study. Thus, it was not possible to determine Samuel Acquah, Christian Bonsu, Stephen Osei-Wusu, Sandra Sowah, possible associations between antimicrobial therapy and resistance Lone Ryste Kildevang Hansen and Julie Hindsberg Nielsen for their patterns. excellent assistance. We conclude that MRSA occurs at low prevalence among S. aureus investigated in this study. 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Graffunder EM and Venezia RA (2002) Risk factors associated with nosocomial methicillin resistant Staphylococcus aureus (MRSA) infection including previous use of antimicrobials. J Antimicrob Chemother 49: 999–1005. PLOS ONE | www.plosone.org 7 February 2014 | Volume 9 | Issue 2 | e89716 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png PLoS ONE Pubmed Central

Molecular Epidemiology and Antimicrobial Susceptibility of Clinical Staphylococcus aureus from Healthcare Institutions in Ghana

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Abstract

The objective of this study was to determine the antimicrobial susceptibility patterns and clonal diversity of clinical Staphylococcus aureus isolates from Ghana. A total of 308 S. aureus isolates from six healthcare institutions located across Northern, Central and Southern Ghana were characterized by antibiotyping, spa typing and PCR detection of Panton Valentine leukocin (PVL) genes. Methicillin-resistant S. aureus (MRSA) were confirmed by PCR detection of mecA gene and further characterized by SCCmec and multi-locus sequence typing (MLST). The prevalence of antimicrobial resistance was below 5% for all agents tested except for penicillin (97%), tetracycline (42%) and erythromycin (6%). Ninety-one spa types were found, with t355 (ST152, 19%), t084 (ST15, 12%) and t314 (ST121, 6%) being the most frequent types. Based on established associations between spa and MLST types, isolates were assigned to 16 clonal complexes (CCs): CC152 (n = 78), CC15 (n = 57), CC121 (n = 39), CC8 (n = 36), CC5 (n = 33), CC1 (n = 29), CC45 (n = 9), CC88 (n = 8), CC30 (n = 4), CC9 (n = 3), CC25 (n = 2), CC97 (n = 2) CC20 (n = 2), CC707 (n = 2), CC7 (n = 3) and CC522 (n = 1). Most isolates (60%) were PVL-positive, especially those belonging to ST152, ST121, ST5, ST15, ST1, ST8, and ST88. Nine (3%) isolates were MRSA belonging to seven distinct clones: ST88-IV (n = 2), ST250-I (n = 2), ST8-IV (n = 1), ST72-V (n = 1), ST789-IV (n = 1), ST2021-V (n = 1), and ST239-III (n = 1). The study confirmed a high frequency of PVL-positive S. aureus in Africa, low prevalence of antimicrobial resistance and high diversity of MRSA lineages in Ghana compared to developed countries and other African countries. The detection of known pandemic MRSA clones in the absence of routine MRSA identification in most Ghanaian clinical microbiology laboratories calls for capacity building to strengthen surveillance and prevent spread of these clones. Citation: Egyir B, Guardabassi L, Sørum M, Nielsen SS, Kolekang A, et al. (2014) Molecular Epidemiology and Antimicrobial Susceptibility of Clinical Staphylococcus aureus from Healthcare Institutions in Ghana. PLoS ONE 9(2): e89716. doi:10.1371/journal.pone.0089716 Editor: J. Ross Fitzgerald, University of Edinburgh, United Kingdom Received October 8, 2013; Accepted January 23, 2014; Published February 25, 2014 Copyright:  2014 Egyir 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. Funding: This study was supported by Antibiotic Drug use, Monitoring and Evaluation of Resistance (ADMER) project funded by the Danish International Development Agency (DANIDA). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interest exist. * E-mail: begyir@sund.ku.dk Africa as recently suggested [5,6]. PVL is associated with SSTI Introduction and severe necrotising pneumonia and has been shown to be a Methicillin-resistant Staphylococcus aureus (MRSA) is a major characteristic feature of community acquired (CA) -MRSA clones concern in clinical medicine due to the importance of b-lactams in disseminated in Europe and Middle East (ST-80), Australia and the therapy of staphylococcal infections and the additional South America (ST30-IV), and United States (ST8-IV, also known morbidity and mortality for MRSA patients compared to patients as USA300) [2,7,8]. infected with methicillin-susceptible S. aureus (MSSA) [1]. Despite The objective of this study was to investigate the antimicrobial the importance of MRSA, MSSA are among the most common susceptibility and clonal diversity of clinical S. aureus isolates from causative agents of bacteraemia and skin and soft tissue infections Ghana. Antimicrobial resistance in S. aureus has previously been (SSTI) [2]. Epidemiological surveillance of MRSA and MSSA is of reported from Ghana with findings of a low MRSA prevalence in importance for the development and implementation of infection nasal swabs from patients and health care workers at the Korle-bu control programmes. Data on S. aureus epidemiology in African Hospital, Accra [9] however, treatment in Ghana is mainly countries are limited and a common trait for MSSA strains from empirical due to a relative lack of appropriate laboratory facilities various African countries seems to be the carriage of the PVL [10] and therefore only few susceptibility data exists and so far no genes: lukS/F-pv at much higher frequencies (.55%) than in the study has investigated the clonal structure of S. aureus in clinical rest of the world (,10%) [2–6]. The high frequency of PVL samples. The study was part of a cooperation program on among human MSSA strains is of special interest since the most Antibiotic Drug use, Monitoring and Evaluation of Resistance successful community associated (CA) MRSA clones share this (ADMER) in Ghana under the Danish Ministry of Foreign Affairs. genetic marker, and could have MSSA ancestors associated with PLOS ONE | www.plosone.org 1 February 2014 | Volume 9 | Issue 2 | e89716 Epidemiology of Staphylococcus aureus in Ghana This program was conceived to strengthen clinical microbiology 15 mg erythromycin, 10 mg norfloxacin, 10 mg gentamicin, 10 mg linezolid, 5 mg rifampicin, 1. 25 mg +23.75 mg trimethoprim- and surveillance of antibiotic resistance, and ultimately to improve awareness of antimicrobial use in Ghana. sulfamethoxazole, and 10 mg fusidic acid (Rosco NeoSenstabs, Taastrup, Denmark). Inducible clindamycin resistance was detected by placing clindamycin and erythromycin 12–20 mm Materials and Methods apart (D-test). Brain Heart Infusion agar supplemented with Ethics Statement teicoplanin (5 mg/L) (Becton Dickinson, Denmark) was used to Ethical clearance was obtained from the University of Ghana screen MRSA isolates for glycopeptides resistance by a spot test; if Medical School Ethical and Protocol Review Board (reference no. 10 or more colonies were detected on these plates, E-tests MS-EI/M.9 - P.3.212010-11). (bioMe´rieux, Marcy I’Etoile, France) were used to determine the minimum inhibitory concentration of vancomycin and teicoplanin [11]. Multidrug resistance (MDR) was defined as resistance to at Bacterial Isolates least three distinct antimicrobial classes or being MRSA [12]. Staphylococcal isolates from clinical specimens were obtained in a prospective cross-sectional-like study between October 2010– June 2012 from six healthcare institution situated at Northern Molecular Typing (Tamale Teaching Hospital), Central (Sunyani Government Molecular characterization of the isolates was done at Statens Hospital) and Southern Ghana (Korle bu Teaching Hospital, Serum Institut (SSI), Denmark. A multiplex PCR was used for Thirty-seven Military Hospital, Ridge Hospital and Legon detection of spa, lukS/F-pv and mecA [13]. spa typing was performed Hospital) (Figure 1). The majority of the isolates (70%) were as described by Harmsen et al. [14]. Using BioNumerics v.6.5 obtained from Korle bu Teaching Hospital, which serves a (Applied Maths, Sint-Martens-Latem, Belgium) with the Ridom population of over 3 million and acts as a major referral health spa server (http://spa.server.ridom.de) plug-in, spa sequences were facility for an estimated population of 24 million people across automatically assigned to spa types and clonal complexes (CCs) Ghana. Presumptive staphylococci identified by colony morphol- based on spa repeats. Multi-Locus Sequence Typing (MLST) [15] ogy at the hospital clinical microbiology laboratories were was done on all MRSA and MSSA isolates whose CC could not be collected and sent to Noguchi Memorial Institute for Medical assigned by the Ridom spa server. Minimum Spanning Tree Research, where they were identified as S. aureus by Gram staining, (MST) based on spa- types was made using BioNumerics V6.5 catalase, tube coagulase and slidex staphplus test (bioMe´rieux, (Applied Maths, Sint-Martens-Latem Belgium). Staphylococcal Marcy l’Etoile, France). Available patient demographic charac- cassette chromosome mec (SCCmec) typing was performed by teristics such as age and sex were retrieved from laboratory multiplex PCR as described previously [16]. records. Statistical Analysis Antimicrobial Susceptibility Testing Distributions of the various genotypes determined in the study Susceptibility testing was carried out by disc diffusion technique (PVL-positivity, spa type, ST and CC) were associated to region, following the European Committee on Antimicrobial Susceptibil- hospital, sex and infection type to determine if specific patterns ity Testing (EUCAST) guidelines (www.eucast.org) using 1U existed. Only genotypes with more than 10 observations were penicillin, 30 mg tetracycline, 30 mg cefoxitin, 2 mg clindamycin, included in statistical analysis. MRSA isolates were not evaluated Figure 1. Origins of the 308 clinical Staphylococcus aureus collected from six hospitals in Ghana, 2010–2012. {Referral Hospitals. 1 2 `Secondary Hospitals. 1Primary Hospitals. SSTI: Skin and Soft Tissue Infections. Other: (Urinary Tract Infection (n = 9); Unknown Infections (n = 14)). doi:10.1371/journal.pone.0089716.g001 PLOS ONE | www.plosone.org 2 February 2014 | Volume 9 | Issue 2 | e89716 Epidemiology of Staphylococcus aureus in Ghana due to the low prevalence of their genotypes. Associations were spatial associations were observed in the distribution of spa- determined using the x test, except for PVL-positivity, which was types, STs and CCs with regard to infection type, sex, region analysed by logistic regression. A significant association was and hospital of origin, Some spatial variations were observed in deemed at p-values ,0.05. the distribution of spa types (e.g. t355 occurred in 34% and 13% isolates from Sunyani Government Hospital and Korle bu Teaching Hospital respectively) but such differences could not Results be proven to be significant. Isolates from Ridge Hospital (n = 1), Of the 903 presumed staphylococci collected from the six Thirty-seven Military Hospital (n = 6) and Tamale Teaching hospitals, 308 (34%) were identified as S. aureus and 595 (66%) as Hospital (n = 12) were excluded from the statistical analysis due coagulase negative staphylococci. S. aureus isolates originated from to low numbers. SSTI (n = 173), bacteraemia (n = 112), and other (urinary tract PVL was detected in 60% (n = 184) of the isolates, mainly infection, n = 9; unknown infections, n = 14) infections (n = 23). S. isolates from SSTIs (57%) and belonging to ST152 (38.5%), aureus was isolated from 143 females and 109 males. Sex origin of ST121 (21%), ST5 (13.5%), ST15 (11%), ST1 (7%), ST8 (3.8%) 56 isolates could not be traced from laboratory records. With and ST88 (2.7%). Genotypes and clinical origins of PVL positive regard to hospital origin, 12 isolates were from Tamale Teaching S. aureus are shown in Table 2. The patterns of PVL varied by Hospital (TTH, Northern Ghana), 53 from Sunyani Government region, with Central regions having a higher chance 2.2 (95% CI: Hospital (SGH, Central Ghana) and 243 from the four hospitals in 1.1–4.2) of seeing PVL-positive S. aureus (p = 0.02) than the Southern Ghana. Details of hospital location (stratified into Southern region. regions of study) and proportions of isolates from clinical infections are shown in Figure 1. None of the clinical laboratories used Discussion methods for MRSA detection and typing, and several pitfalls were recognized in routine microbiological procedures (e.g. poor This study fills an important gap in the knowledge of the identification to species/genus level, and low compliance with epidemiology of S. aureus in Ghana. As such, the study international standards for susceptibility testing). contributes to the current knowledge of the diversity and The highest prevalence of resistance was for penicillin (97%), population structure of this important bacterial pathogen at the followed by tetracycline (42%) and erythromycin (6%). Lower global level. Ghana and several other African countries have so percentages of resistance were observed for clindamycin (5%), far been black spots on the map due to lack of established norfloxacin (4%), trimethoprim-sulphamethoxazole (4%), genta- national surveillance programmes and adequate clinical micro- micin (3%), cefoxitin (3%) and fusidic acid (2%). Inducible biology infrastructure [10,17]. Our results show that the most clindamycin resistance was detected among seven (2%) isolates. common spa types among MSSA isolates are t355 (ST152) and Twenty-nine isolates (9%) were MDR, of which 9 (3%) were t084 (ST15). The spa types were previously found to be confirmed mecA positive MRSA. Details of MDR isolates have predominant among S. aureus isolates from asymptomatic nasal been shown in Table 1. MRSA isolates were susceptible to carriers at Korle bu, the largest Teaching Hospital in Ghana vancomycin and teicoplanin. Most of the MSSA isolates (88%, [9], suggesting that they are well established in the human 264/299) were resistant to penicillin (n = 154), penicillin and population of this country. In another African study, t084 tetracycline (n = 99) and penicillin and trimethoprim-sulphameth- (ST15) was also reported as one of the most frequent spa types oxazole (n = 11). All isolates were susceptible to linezolid and among S. aureus isolated from seven tertiary hospitals located in rifampicin while three isolates (1%) were susceptible to all five major African towns [3]. PVL-positive ST152 (t355) is also antimicrobial drugs tested. widely distributed in African countries [4,5] and its frequent High genetic diversity was observed by spa typing, as indicated recovery from SSTI is consistent with studies in other countries by the recovery of 91 spa types among all isolates tested. The most [4,18]. Other PVL-positive MSSA lineages found in this study common spa types were t355 (19%), t084 (12%), t314 (6%) and such as ST121, ST30, ST15 and ST5 have also been reported t311 (5%). Fifty-six spa types were singletons and eight new spa elsewhere in Africa [19]. The observed high prevalence (60%) types were detected: t10809 (ST88), t10810 (ST88), t10811 (ST8), of PVL appears to be a distinguishing genetic trait of African t10828 (ST152), t10833 (ST152), t10836 (ST1), t10837 (ST1) and MSSA [3,4,6] compared to USA, Asia and Europe, where this t10838 (ST30). A minimum spanning tree, including spa types and virulence factor is uncommon in MSSA [2,19,20]. This finding clonal complexes of the 308 isolates is shown in Figure 2. was correlated to the high frequency of PVL-positive ST152, which is a likely ancestor of the CA-MRSA ST152-V clone Based on spa typing, isolates (n = 308) were assigned to 16 MLST clonal complexes: CC152 (n = 78), CC15 (n = 57), CC121 circulating in certain European regions, especially the Balkan (n = 39), CC8 (n = 36), CC5 (n = 33), CC1 (n = 29), CC45 (n = 9), area [21,22]. CC88 (n = 8), CC30 (n = 4), CC9 (n = 3), CC7 (n = 3), CC25 The nine MRSA isolates belonged to seven unrelated spa types (n = 2), CC97 (n = 2) CC20 (n = 2), CC707 (n = 2), and CC522 and STs harbouring four different SCCmec types (Table 1), (n = 1). MRSA isolates belonged to ST88-IV (n = 2), ST8-IV indicating high clonal diversity. Some of the MRSA lineages (n = 1), ST789-IV (n = 1), ST72-V (n = 1) ST2021-V (n = 1), identified in this study are widely distributed worldwide: ST239-III ST250-I (n = 2), and ST239-III (n = 1). Two of them were PVL- is a pandemic clone prevalent in Europe, Asia and South Africa positive and belonged to t121 (ST8) and t547 (ST789) (Table 1). [23–25] and ST789-IV is a single locus variant of the ST7 clone The most common MSSA lineages were ST152 (27%) and ST15 frequently reported in Asia [25]. ST88-IV, ST8-IV and ST72-V (18%). have been previously reported among inpatients and staff at Five spa types (t084, t127, t311, t314 and t355), six STs (ST1, Korle-bu Hospital in Ghana [9] and in communities and hospitals ST5, ST8, ST15, ST121 and ST152) and six CCs (CC1, CC5, in other African countries [26,27]. MRSA ST88 has been reported CC8, CC15, CC121 and CC152) were included in the sporadically in some European countries like Portugal [28] and statistical analysis. Surprisingly, spa type t311 occurred less Sweden [29]. ST8-IV MRSA (spa type t121, PVL+) found in this frequently among females (0.3%) than among males (2%) and study is related to the epidemic MRSA ST8-IV (USA300) clone in those of unknown sex (2.3%) (p = 0.0016). No other clinical or the USA [2]. Other African studies have reported this ST8-IV PLOS ONE | www.plosone.org 3 February 2014 | Volume 9 | Issue 2 | e89716 Epidemiology of Staphylococcus aureus in Ghana Table 1. Origins and characteristics of 29 multi-drug resistant (MDR) Staphylococcus aureus isolated from healthcare institutions in Ghana, 2010–2012. a b c ID Hospital Infection CC ST spa type SCCmec PVL Antibiotype MRSA 5016 KB SSTI CC1 ST72 t537 V 2 Fox, Pen, Tet 744 KB Blood CC8 ST2021 t024 V 2 Fox, Pen, Tet 2244 KB Blood CC8 ST239 t037 III 2 Fox, Pen,Tet, Fuc, Gen, Cli, Ery, 3464 KB Blood CC8 ST8 t121 IV + Fox, Pen, Nor, Cli, Ery 2207 KB SSTI CC8 ST250 t928 I 2 Fox, Pen, Tet, Gen, Nor, Cli, Ery 2224 KB SSTI CC8 ST250 t928 I 2 Fox, Pen, Tet, Gen, Nor, Cli, Ery 44 SGH Unknown CC88 ST88 t186 IV 2 Fox, Pen, Tet AU81 SGH SSTI CC88 ST88 t186 IV 2 Fox, Pen, Tet 11087 KB UTI CC152 ST789 t547 IV + Fox, Pen, Tet, Nor MSSA 2639 KB Blood CC1 ST1 t7835 NA + Pen, Tet, Cli, Ery AU93 SGH SSTI CC1 ST1 t559 NA + Pen, Tet, Fuc A6 KB Unknown CC5 ST5 t311 NA + Pen, Tet, Fuc 5095 KB SSTI CC5 ST5 t071 NA + Pen, Tet, Gen, TMS, Fuc, Nor, Cli, Ery T2845 TTH SSTI CC8 ST8 t451 NA + Pen, Tet, TMS 1455 KB SSTI CC9 ST9 t2700 NA 2 Pen, Gen, Cli, Ery 1050 KB Blood CC15 ST15 t084 NA + Pen, Tet, Gen 2320 KB Blood CC45 ST508 t635 NA 2 Pen, Cli, Ery 1548 KB SSTI CC88 ST88 t10809 NA + Pen, Tet, Nor, Cli, Ery 5270 KB SSTI CC88 ST88 t10810 NA + Pen, Tet, Nor, Cli, Ery NAB KB SSTI CC121 ST121 t213 NA _ Pen, Tet, Fuc 3209 KB Blood CC121 ST121 t091 NA _ Pen, Tet, Nor, Gen, Cli, Ery 2437 KB Blood CC121 ST121 t091 NA _ Pen, Tet, Nor 5293 KB Blood CC121 ST121 t314 NA + Pen, Tet, Nor 5775 KB Blood CC121 ST121 t314 NA + Pen, Tet, Nor 3984 KB SSTI CC152 ST152 t1299 NA + Pen, Tet, Cli, Ery 1544 KB SSTI CC152 ST152 t355 NA + Pen, Tet, Cli, Ery 4836 KB SSTI CC152 ST152 t355 NA + Pen, Tet, Cli, Ery 112242 MH SSTI CC152 ST152 t355 NA + Pen, Tet, Ery A71 SGH Blood CC152 ST152 t355 NA + Pen, Tet, Fuc ST: Sequence Type; CC: Clonal Complex; SCC: Staphylococcal Cassette Chromosome; PVL: Panton- Valentine leukocidin. KB: Korle bu Teaching Hospital; SGH: Sunyani Government Hospital; TTH: Tamale Teaching Hospital; MH: Military Hospital. b c SSTI: Skin and Soft Tissue Infection; UTI: Urinary Tract Infection; TMS: trimethoprim-sulphamethoxazole. Pen: penicillin; Fox: cefoxitin; Tet: tetracycline; Nor: norfloxacin; Gen: gentamicin; Fuc: Fucidic acid; Cli: clindamycin; Ery: erythromycin. doi:10.1371/journal.pone.0089716.t001 MRSA (spa type t121, PVL+) strain in communities and hospitals The prevalence of antimicrobial resistance in clinical S. aureus [26,30]. ST250-I, also referred to as the ‘‘Archaic clone’’, differs isolates from Ghana was generally low. Other African studies have from ST8 by a point mutation in the yqiL gene and is related to reported similar levels of resistance to penicillin (86%–93%) and ST247-I (Iberian clone), a major clone isolated in European tetracycline (28%–48%) but higher levels of resistance to hospitals [7,31]. ST72 has been reported as a major MRSA clone sulphonamides (22%–68%) compared to this study [3,4,9]. from communities in Australia [32] and as MSSA in Nigeria and Comparatively, the prevalence of MRSA (3%) was lower than Gabon [6,27]. The least known MRSA lineage found in this study those reported in other African countries such as Nigeria (20%) was ST2021-V, which to the best of our knowledge has previously [27], Algeria (45%) [33] and in a multicenter study (15%) been reported in a single isolate from Nigeria (www.mlst.net; involving five major African towns [26]. The low MRSA th accessed on: 4 April 2013). Although ST5, ST30 and ST80 frequency reported in this study could be attributed to the low MRSA have been described in several African and other countries consumption of antimicrobial agents such as fluoroquinolones and around the world [26,27], none of these clones were detected third generation cephalosporins in Ghana, because they are among clinical MRSA isolates in Ghana. PVL-positive ST5 and expensive and are usually prescribed for acute infections [10]. ST30 were however detected among MSSA isolates (Table 2), Usage of the afore-mentioned antimicrobial agents has been indicating that these two S. aureus lineages are widespread in shown to correlate with an increase in MRSA prevalence [34–36]. African countries, even though acquisition of methicillin resistance The observed MRSA prevalence among clinical isolates in Ghana seems to be confined to some countries. is similar to those reported in European countries with low MRSA PLOS ONE | www.plosone.org 4 February 2014 | Volume 9 | Issue 2 | e89716 Epidemiology of Staphylococcus aureus in Ghana Figure 2. Minimum spanning tree of 308 clinical Staphylococcus aureus isolates from healthcare institutions in Ghana. Nodes indicate spa types and their size shows the relative number of isolates for each spa type. Numbers of frequent (three or more) spa types have been shown. Every colour represents a distinct clonal complex. doi:10.1371/journal.pone.0089716.g002 Table 2. Clonal complex (CC), multi-locus sequence type (ST), spa type and clinical origin of 184 Staphylooccus aureus harbouring Panton-Valentine leukocidin (PVL) genes isolated in Ghana, 2010–2012. STs and CCs were inferred from spa types. Clinical origin, N (%) CC ST spa type (N) Bacteraemia SSTI Other* Total N=65 N = 104 N=15 N = 184 CC1 ST1 t127 (3), t1931 (3), t693 (1), t559 (1), t10836 (1), 6 (9.2) 7 (6.7) 0 (0.0) 13 (7.0) t114 (1), t922 (1), t934 (1), t7835 (1) CC5 ST5 t071 (9) t311 (9), t002 (5), t105 (1) 12 (18.5) 11 (10.6) 1 (6.7) 24 (13.0) CC8 ST8 t1476 (3), t024 (1), t451 (1), t064 (1), ut121(1) 2 (3.0) 5 (4.8) 0 (0.0) 7 (3.8) CC15 ST15 t084 (19), t5534 (1), t774 (1) 7 (10.8) 13 (12.5) 1 (6.7) 21 (11.4) CC25 ST25 t401 (1) 1 (1.5) 0 (0.0) 0 (0.0) 1 (0.5) CC30 ST30 t10838 (1),t363 (1) 0 (0.0) 2 (1.9) 0 (0.0) 2 (1.1) CC88 ST88 t2393 (2) t3202 (1), t10809 (1), t10810 (1) 1 (1.5) 4 (3.8) 0 (0.0) 5 (2.7) CC121 ST121 t314 (15), t2304 (9), t159 (5), t645 (5), t1077(1), t7002(1) 14 (21.5) 21 (20.2) 1 (6.7) 36 (19.6) CC152 ST152 t355 (57), t4690 (3), t1096 (2), t1299 (2), Singletons (11) 22 (34.0) 41 (39.4) 12 (80.0) 75 (40.8) SSTI: Skin and Soft Tissue Infection;*Other: UTI: Urinary Tract Infection (n = 5: spa types t355 (3), t547(1) and t5534 (1); Unknown (n = 10; spa types: t311 (1), t645 (1), t4690 (1), t355 (7) Other spa types associated with CC152: t454, t458, t5268, ut547, t1123, t1172, t5047, t7011, t8821, t10828, and t10833uMRSA. doi:10.1371/journal.pone.0089716.t002 PLOS ONE | www.plosone.org 5 February 2014 | Volume 9 | Issue 2 | e89716 Epidemiology of Staphylococcus aureus in Ghana prevalence, such as the Scandinavian countries and The Nether- and poor performance standards in most clinical microbiology lands [37]. laboratories in Ghana, highlighting the need for infrastructures Some apparent geographical variations in clonal distribution to support national antimicrobial policies and surveillance were observed, but the low number of isolates obtained from the capacity. Northern region made comparisons between hospitals or regions meaningless. The clinical information on the 308 S. aureus included Acknowledgments in the study varied in quality due to incompleteness of the patient The directors of hospitals are thanked for their support to the study. The records collected from the various hospital clinical laboratories authors are grateful to Dr. Jesper Larsen, Mr Michael Olu-Taiwoo, Mr involved in the study. Thus, it was not possible to determine Samuel Acquah, Christian Bonsu, Stephen Osei-Wusu, Sandra Sowah, possible associations between antimicrobial therapy and resistance Lone Ryste Kildevang Hansen and Julie Hindsberg Nielsen for their patterns. excellent assistance. We conclude that MRSA occurs at low prevalence among S. aureus investigated in this study. 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Graffunder EM and Venezia RA (2002) Risk factors associated with nosocomial methicillin resistant Staphylococcus aureus (MRSA) infection including previous use of antimicrobials. J Antimicrob Chemother 49: 999–1005. PLOS ONE | www.plosone.org 7 February 2014 | Volume 9 | Issue 2 | e89716

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