The dominant methicillinresistant ... - Wiley Online Library

ORIGINAL ARTICLE

EPIDEMIOLOGY

The dominant methicillin-resistant Staphylococcus aureus clone from hospitals in Cape Town has an unusual genotype: ST612 M. J. Jansen van Rensburg1, V. Eliya Madikane1,2, A. Whitelaw1,2,3, M. Chachage1, S. Haffejee2 and B. Gay Elisha1,2,3 1) Division of Medical Microbiology, University of Cape Town, 2) National Health Laboratory Service and 3) National Institute of Communicable Diseases, National Health Laboratory Service, University of Cape Town, Cape Town, South Africa

Abstract There is currently limited information available on the molecular epidemiology of methicillin-resistant Staphylococcus aureus (MRSA) in South Africa. A molecular characterization of 100 MRSA from five hospitals in Cape Town was carried out in this study. The strains were separated into six clusters by pulsed-field gel electrophoresis, indicating transmission of MRSA between local hospitals. None of the strains carried the Panton-Valentine Leukocidin gene. SCCmec typing, multilocus sequence typing and spa typing were used to further characterize the MRSA. Three clones corresponded to frequently described pandemic clones: ST239-MRSA-III, ST36-MRSA-II and ST5-MRSA-I. ST239-MRSA-III and ST36-MRSA-II were minor clones and collectively accounted for 16% of the isolates. ST5-MRSA-I was the second-most prevalent clone and accounted for 37% of the isolates. The dominant local clone was the infrequently described ST612-MRSA-IV (44% of isolates), which has only been described in South Africa and Australia. Keywords: Methicillin-resistant Staphylococcus aureus, MLST, SCCmec, ST612 Original Submission: 16 April 2010; Revised Submission: 30 August 2010; Accepted: 30 August 2010 Editor: G. Lina Article published online: 20 September 2010 Clin Microbiol Infect 2011; 17: 785–792 10.1111/j.1469-0691.2010.03373.x Corresponding author: B. Gay Elisha, Division of Medical Microbiology, Faculty of Health Sciences, University of Cape Town, Anzio Road, Observatory 7925, South Africa E-mail: [email protected]

Introduction Methicillin-resistant S. aureus (MRSA) has become a major public health problem worldwide [1,2], and South Africa is no exception. The proportion of clinical S. aureus isolates that are methicillin-resistant in South Africa ranges from 29% to 46% [3,4]. Methicillin-resistance is conferred by mecA, which is contained in a large mobile genetic element called the staphylococcal cassette chromosome mec (SCCmec) [2,5]. The SCCmec element has been acquired on numerous occasions by a limited number of S. aureus lineages or clonal complexes (CCs), including CC5, CC8, CC22, CC30 and CC45 [6,7]. The global prevalence of MRSA is due to the dissemination of pandemic clones, and SCCmec element capture by local methicillin-susceptible S. aureus (MSSA) [6–8]. Traditionally, MRSA is regarded as a nosocomial pathogen; however, in recent decades, it has also emerged in the

community [1,2,5]. Although the differences between hospital-acquired MRSA (HA-MRSA) and community-acquired MRSA (CA-MRSA) have become less distinct, the former commonly contain SCCmec types I–III, and are often resistant to non-b-lactam antibiotics. On the other hand, CAMRSA isolates typically contain SCCmec types IV, VI and VII, often carry the Panton-Valentine Leukocidin (pvl) gene, and are typically susceptible to non-b-lactam antibiotics [2,5]. Notwithstanding the characterization of MRSA from the KwaZulu-Natal province [9,10], there is a paucity of comprehensive data on the molecular epidemiology of MRSA in South Africa. This report describes the molecular epidemiology of MRSA circulating in hospitals in Cape Town, in the Western Cape province of South Africa.

Materials and Methods Bacterial isolates

One hundred MRSA isolates, without duplication, obtained between January 2007 and December 2008, were included in the study. This represented 13.6% of MRSA isolates identified in the laboratory over the study period. The isolates were cultured from pus and pus swabs (n = 64), urine

ª2010 The Authors Clinical Microbiology and Infection ª2010 European Society of Clinical Microbiology and Infectious Diseases

786

Clinical Microbiology and Infection, Volume 17 Number 5, May 2011

(n = 9), respiratory tract specimens (n = 13), blood (n = 7) and central venous catheter tips (n = 7). The specimen types were broadly representative of those from which MRSA were isolated during the study period and were obtained from patients in any one of five hospitals in metropolitan Cape Town: Groote Schuur Hospital (GSH, n = 51), Mowbray Maternity Hospital (MMH, n = 19), Red Cross War Memorial Children’s Hospital (RCCH, n = 21), Victoria Hospital (VH, n = 4) and University of Cape Town Private Hospital (UCTPH, n = 5). The number of isolates from each hospital was representative of the proportions isolated from patients at each hospital. GSH, RCCH and UCTPH are tertiary level hospitals with bed capacities of 893, 240 and 112, respectively. These hospitals offer a range of clinical services, including general medical and surgical, trauma and emergency, as well as subspecialist medical and surgical services. VH (158 beds) is a secondary level hospital offering emergency, trauma and general medical and surgical services. Although MMH (179 beds) is a secondary level maternity hospital, tertiary neonatal care is offered. The study isolates were obtained from patients in a range of wards, including outpatient clinics (n = 15), emergency services (n = 4), medical (n = 16), surgical (n = 23), intensive care units (n = 20), and obstetrics and gynaecology (n = 15). No ward was specified for seven of the samples. The National Health Laboratory Service microbiology laboratory at GSH serves all five hospitals. Definition of community-acquired MRSA

Although the purpose of the study was not to compare the epidemiology of CA-MRSA with that of HA-MRSA, a rudimentary approach was used to classify the isolates. Admission histories of all patients were reviewed using electronic admission records. These records did not include a history of prior admission to private health care facilities, nursing facilities, or health care facilities outside the city of Cape Town. MRSA isolated within 48 h of admission to a hospital, from patients with no record of prior admission to a health care facility, or contact with an outpatient clinic within the previous 12 months, was classified as CA-MRSA. All others were classified as HA-MRSA. Isolate identification and antibiotic susceptibility testing

Isolates were identified either by the production of DNAse, or on the VITEK 2 (bioMe´rieux, La Balme-les-Grottes, France). Antimicrobial susceptibility testing was performed using disc diffusion or the VITEK 2 system. Zone sizes or VITEK 2 MIC results were interpreted using Clinical and Laboratory Standards Institute breakpoints [11,12], as well as the Advanced Expert System of the VITEK 2.

CMI

Pulsed-field gel electrophoresis

Pulsed-field gel electrophoresis (PFGE) profiles were obtained using the method described by Reed et al. [13] with the following modifications. Bacterial cell suspensions were standardized to an OD600 reading between 0.825 and 0.875. Agarose plugs (width, 5mm; height, 2mm; and thickness, 1mm) were prepared with 2% SeaKem Gold Plug Agarose (Cambrex Bio Science Rockland, Inc., Rockland, ME, USA). Electrophoresis was carried out using a CHEF-DRII GeneNavigator (Amersham Biosciences, Fairfield, CT, USA) at a pulse time of 5–60 s for 22 h at 200 V with 0.5· TBE maintained at 14C. Digital images were analysed using GelCompar II (version 4.6) (Applied Maths BVBA, Sint-Martens-Latem, Belgium). The images were normalized in comparison to the profile of S. aureus NCTC8325. Only fragments within the normalized region of each gel were included in the analysis [14]. A dendrogram was created with the cluster analysis function using the Dice similarity coefficient and unweighted pair-group method, using averages (UPGMA) with a position tolerance setting of 1% and an optimization value of 0.5%. PFGE clusters were defined by a similarity level of at least 80% [13,14]. Detection of SCCmec types

Genomic DNA was isolated using the QIAamp DNA Mini kit (Qiagen, Valencia, CA, USA). SCCmec types were determined by multiplex PCR [15]. Where necessary, atypical amplification patterns were resolved using primers described by Kondo et al. [16] to determine the combination of mec class and ccr allotype present. Detection of pvl

The presence of pvl was determined by PCR using primers luk-PV-1 and luk-PV-2 as described by Lina et al. [17]. Multi-locus sequence typing and spa typing

Multilocus sequence typing (MLST) and spa typing were carried out as previously described [18,19]. PCR products were purified using the MinElute Gel Extraction kit (Qiagen) and sequenced at the Central Analytical Facility at the University of Stellenbosch. MLST sequence types (STs) were determined using the online database (http://www.mlst.net) while spa types were determined with Ridom StaphType (version 1.5.21) (Ridom GmbH, Wu¨rzburg, Germany).

Results PFGE profiles

The MRSA segregated into six PFGE clusters (A–F) and eight sporadic isolates (Fig. 1). Sixty-eight per cent of the strains

ª2010 The Authors Clinical Microbiology and Infection ª2010 European Society of Clinical Microbiology and Infectious Diseases, CMI, 17, 785–792

CMI

Jansen van Rensburg et al. MRSA from hospitals in Cape Town

787

FIG. 1. Pulsed-field gel electrophoresis profiles and dendrogram showing the level of similarity between 100 methicillin-resistant Staphylococcus aureus strains. Six clusters were identified and are indicated alongside each profile. Strain designations used in the text for isolates selected for multilocus sequence typing and spa typing are indicated in brackets. Putative community-acquired isolates are indicated by *.

was almost equally distributed between clusters C (n = 35) and E (n = 33). The remaining MRSA were assigned to clusters A (n = 4), B (n = 2), D (n = 7) and F (n = 11). When

clusters were stratified by hospital, strains from cluster E were identified in all of the hospitals (Table 1). Clusters C and D included strains from three hospitals.


788


CMI

FIG. 1. (Continued)

SCCmec typing, MLST and spa typing

SCCmec types I and IV were identified in 37 strains (clusters B and C) and 46 strains (clusters D and E). SCCmec types II

(cluster F) and III (cluster A) were observed in 11 and 4 strains, respectively (Table 1). SCCmec type IV was identified in six sporadic isolates. SCCmec type II was identified in one



CMI

789

TABLE 1. Stratification of pulsedDistribution of strains across hospitals [no. (%)]

field gel electrophoresis (PFGE) clusters by hospital

PFGE cluster (no.)

SCCmeca type

GSHb

RCCH

MMH

VH

UCTPH

A (4) B (2) C (35) D (7) E (33) F (11)

III I I IV IV II

2 1 13 4 19 8

2 – 4 2 9 –

– – 18 (51.4) – 1 (3.0) –

– – – 1 (14.3) 3 (9.1) –

– 1 (50) – – 1 (3.0) 3 (27.3)

(50) (50) (37.1) (57.1) (57.6) (72.7)

(50) (11.4) (28.6) (27.3)

a

SCCmec, staphylococcal cassette chromosome mec. GSH, Groote Schuur Hospital; RCCH, Red Cross War Memorial Children’s Hospital; MMH, Mowbray Maternity Hospital; VH, Victoria Hospital; UCTPH, University of Cape Town Private Hospital.

b

NT, t3092, and ST22-MRSA-IV, t032, while the PFGE outlier, designated strain S8, corresponded to ST650 and was a single locus variant (SLV) of ST5 (varying at the arcC locus), t002, with SCCmec type IV.

of the remaining sporadic isolates, whereas the other was non-typable (NT). MLST and spa typing were used to further characterize the isolates. For these studies, a strain representative of each of the PFGE clusters A, B, C, D and F was chosen. As varying levels of genetic similarity were observed within cluster E, three strains were selected from this cluster. All sporadic isolates were included in these investigations. The STs and other relevant properties of the MRSA are presented in Table 2. Representatives of clusters A, B, C and F corresponded to frequently described pandemic clones. MRSA from cluster A corresponded to ST239-MRSA-III, spa type t037; B and C corresponded to ST5-MRSA-I, t045, whereas ST36-MRSA-II was identified for F (t012) and one sporadic isolate (t021). Representative strains from clusters D and E, and four sporadic isolates, were consistent with the infrequently described ST612-MRSA-IV clone. A number of spa types were detected in the ST612-MRSA-IV strains (Table 2). Two of the remaining sporadic isolates equated with ST72-MRSA-

Antimicrobial susceptibility profiles

There were striking differences between the antimicrobial susceptibility profiles of the clones (Table 3). Resistance to gentamicin, co-trimoxazole, ciprofloxacin and rifampicin was widespread in ST612-MRSA-IV strains, with 43 strains (97.73%) resistant to three or more classes of antibiotics in addition to the b-lactams (Table 3). In contrast, the ST5MRSA-I strains were generally susceptible to non-b-lactam antibiotics, with the exception of erythromycin and clindamycin (Table 3). The detection of pvl

Ten isolates were classified as CA-MRSA: one from MMH, six from RCCH and three from GSH. These isolates were cultured from pus swabs (n = 6), urine (n = 3) and sputum

TABLE 2. Characteristics of MRSA strains selected for multilocus sequence typing and spa typing MRSA designationa

PFGEb cluster (no. isolates)

SCCmecc type

Antimicrobial resistance profiled

MLST (ST; CC)e

spa type

A1 B1 C1 D1 E1 E2 E3 F1 S1 S2 S3 S4 S5 S6 S7 S8

A (4) B (2) C (35) D (7) E (33) E (33) E (33) F (11) Sporadic Sporadic Sporadic Sporadic Sporadic Sporadic Sporadic Sporadic

III I I IV IV IV IV II IV Non-typable II IV IV IV IV IV

PEN, PEN, PEN, PEN, PEN, PEN, PEN, PEN, PEN, PEN, PEN, PEN, PEN, PEN, PEN, PEN,

239; 8 5; 5 5; 5 612; 8 612; 8 612; 8 612; 8 36; 30 612; 8 72; 8 36; 30 612; 8 612; 8 612; 8 22; 22 ST650f; 5

t037 t045 t045 t064 t1443 t2196 t1443 t012 t1443 t3092 t021 t1257 t064 t064 t032 t002

isolate isolate isolate isolate isolate isolate isolate isolate

OXA, OXA, OXA OXA, OXA, OXA, OXA, OXA, OXA, OXA, OXA, OXA, OXA, OXA, OXA, OXA

ERY, CLI, SXT, CIP, GEN ERY, CLI, GEN ERY, CLI, RIF, SXT, GEN RIF, SXT, CIP, GEN ERY, CLI, RIF, SXT, CIP, GEN RIF, SXT, CIP, GEN ERY, CLI, CIP RIF, SXT, CIP, GEN ERY, CLI ERY, CLI, CIP, GEN ERY, CLI, RIF, SXT, CIP, GEN RIF, SXT, GEN RIF, SXT, GEN ERY, CLI, CIP

a

Strain designations of representative isolates as indicated in figure and text. PFGE, pulsed-field gel electrophoresis. c SCCmec, staphylococcal cassette chromosome mec. d PEN, penicillin; OXA, oxacillin; ERY, erythromycin; CLI, clindamycin; RIF, rifampicin; SXT, co-trimoxazole; CIP, ciprofloxacin; GEN, gentamicin. e MLST, multilocus sequence typing; ST, sequence type; CC, clonal complex. f Single locus variant of ST5 (varying at the arcC locus). b


790


CMI

TABLE 3. Antimicrobial susceptibility profiles of predominant clonal types Antimicrobial agents (no. (%) resistant) Clonal type

PFGE cluster (no. isolates)

ERY

CLI

RIF

SXT

CIP

GEN

ST239-MRSA-III ST5-MRSA-I ST612-MRSA-IV ST36-MRSA-II

A (4) B, C (2, 35) D, E (7, 33) F (11)

4 34 18 11

4 34 16 11

0 1 (2.7) 40 (100) 1 (9.1)

4 (100) 1 (2.7) 38 (95) 0

4 1 29 10

4 11 36 3

(100) (91.9) (45) (100)

(100) (91.9) (40) (100)

(100) (2.7) (72.5) (90.9)

(100) (29.7) (90) (27.3)

ERY, erythromycin; CLI, clindamycin; RIF, rifampicin; SXT, co-trimoxazole; CIP, ciprofloxacin; GEN, gentamicin.

(n = 1). Three and seven of the putative CA-MRSAs corresponded to ST5-MRSA-I and ST612-MRSA-IV, respectively. The pvl gene was not detected in any of the 100 isolates.

Discussion To the best of our knowledge, this is the first study describing the molecular characteristics of MRSA from hospitals in Cape Town, South Africa. One hundred MRSA isolates from patients in five hospitals, including ten possible CA-MRSA, were characterized in this study. The distribution of PFGE clusters, SCCmec types and STs did not differ between the HA-MRSA and putative CA-MRSA, and the pvl gene was not detected in any of the isolates. This was consistent with previous studies in which this gene was either infrequently or not detected in South African MRSA strains [10,20,21]. The MRSA segregated into six PFGE clusters and eight sporadic isolates (Table 1 and Fig. 1). Each of the clusters comprised strains from two or more hospitals, demonstrating transmission of strains within and between hospitals. Clinicians commonly render clinical services at more than one hospital, and this may represent one route by which MRSA strains have disseminated across hospitals. Additionally, the transfer of patients between hospitals is common practice and almost certainly serves as a second means of spread. Representatives of the PFGE clusters, and the sporadic isolates, were characterized further using MLST and spa typing. Four clonal types, ST239-MRSA-III, ST36-MRSA-II, ST5MRSA-I and ST612-MRSA-IV, accounted for the majority of the strains. The remaining three sporadic isolates designated S2, S7 and S8 corresponded to: ST72-MRSA-NT, t3092, ST22-MRSA-IV, t032, and ST650-MRSA-IV, t002. With respect to sporadic isolates S2 and S8: ST72-MRSA isolates have been described in Europe, the United States of America and Brazil; in these countries the lineage is typically associated with SCCmec type IV [22,23]. ST72 has also become prevalent in Korea, where isolates containing either SCCmec type II or IV have been described [24]. S8, ST650MRSA, contained SCCmec type IV and was susceptible to all non-b-lactam antibiotics, which are characteristics typical of

CA-MRSA, suggesting that this strain could have originated in the community. However, the available admissions data suggested that infection was nosocomially acquired. ST239-MRSA-III, which has been assigned numerous monikers, including Brazilian/Hungarian, was a major pandemic clone in Europe and South America during the 1990s [25] and the second major clone recovered from health care facilities in the KwaZulu-Natal province, South Africa, between 2001 and 2003 [10]. However, it was the smallest clonal type identified in this study, including only four strains. Based on recent studies on the population structure of ST239-MRSAIII [25], this clone was probably imported into South Africa. The representative of 11 MRSA isolates in cluster F (F1, Fig. 1), and one sporadic isolate, S3, corresponded to ST36MRSA-II. This clone, also called EMRSA-16, has been reported in South Africa [4,20] and was one of the predominant nosocomial clones circulating in the United Kingdom (UK) [26]. It has been proposed that the spread of EMRSA-16 to Spain and Malta resulted from travel between the countries [27]. With a history of emigration from the UK to South Africa, and tourism between the two countries, it is likely that EMRSA-16 was imported into South Africa from the UK. The sporadic isolate, ST22-MRSA-IV, identified in one local hospital may have travelled a similar route. This clone, also known as EMRSA-15, has spread worldwide since it was first reported as epidemic in hospitals in the UK [2,26]. ST5-MRSA-I (EMRSA-3), a pandemic clone described worldwide [2], was the second-most prevalent clone in Cape Town hospitals. Although ST5-MRSA strains have been identified previously in South Africa, they contained SCCmec types III or IV [9,10]. To the best of our knowledge, this is only the second report of ST5-MRSA-I in South Africa [8– 10]. It is interesting to consider the origin of local ST5MRSA-I strains in the light of the study reported by Nu¨bel et al. [8]. Their study suggested that geographical dispersal of ST5-MRSA has been limited, and this clone has emerged on several occasions in geographically distinct regions. While it is possible that local ST5-MRSA-I strains were imported from another region, the possibility that the clone emerged following the acquisition of SCCmec type I by MSSA in hospitals in Cape Town should also be considered.


CMI


The infrequently described ST612-MRSA-IV was the dominant clone circulating in hospitals in Cape Town. To the best of our knowledge, this clone, a member of CC8, has been described only in South Africa and Australia, and then only twice in each country [20, http://www.mlst.net]. That the geographical scatter of ST612 may be limited is supported by recent studies [8,28,29], which showed that certain S. aureus lineages are largely endemic to particular regions. Given the degree of genetic variation revealed by PFGE, in addition to the number of spa types detected for ST612-MRSA-IV strains, it is possible to suggest that this is an old local clone that has accumulated genetic variation over time and undergone clonal expansion. All of the local ST612-MRSA-IV strains were resistant to a number of antibiotics, including rifampicin and co-trimoxazole. A similar level of resistance to these antibiotics was observed in MRSA strains from hospitals in the KwaZuluNatal and Gauteng provinces [10,30]. Interestingly, the majority of resistant isolates from the former study had STs closely related to ST612 and also carried SCCmec type IV [10]. It is tempting to suggest that these MRSAs are best fitted to the current South African environment where rifampicin is commonly prescribed for the treatment of tuberculosis, and co-trimoxazole for the prophylaxis or treatment of Pneumocystis infections in HIV-positive persons. It is interesting to consider the relatedness of local ST612-MRSA-IV to the previously described isolates from South Africa and Australia. No clinical data were available for the previously described ST612-MRSA-IV. Laboratory information showed that they were resistant to rifampicin. An investigation of the mechanism of resistance to rifampicin in these strains, and nine ST612-MRSA-IV identified in this study, revealed that all isolates had two amino acid substitutions (H481N, I527M), which are associated with high level rifampicin resistance (data not shown). Further, two synonymous single nucleotide polymorphisms were identified in the rifampicin-resistance determining region in all 13 isolates. Additionally, the two ST612-MRSA-IV previously isolated in South Africa, and one of the Australian strains, had spa type t064, which was identified in three of the local isolates. Taken together the data suggest that the isolates from Cape Town may share a common ancestor with previously described ST612-MRSA-IV isolates, but further studies are necessary for confirmation.

Acknowledgements We thank Professor Hermıńia de Lencastre for providing S. aureus strains COL, BK2464, ANS46, MW2 and HDE288,

791

and Professor Keiichi Hiramatsu for providing WIS, which were used as control strains for SCCmec typing. We also thank Professor Richard Goering, Dr Frances O’Brien and Julie Pearson for providing the previously described ST612MRSA-IV strains. Aspects of this work were presented at the 3rd Joint Federation of Infectious Diseases Societies of Southern Africa Congress, 20–23 August 2009, Sun City, South Africa.

Transparency Declaration This study was supported by grants from the University of Cape Town and the National Health Laboratory Service. MJJvR was supported by the University of Cape Town, the National Research Foundation, the Ernst and Ethel Eriksen Trust and the Stella and Paul Loewenstein Charitable and Educational Trust. The authors have no conflicts of interest to declare.

References 1. Witte W, Cuny C, Klare I, Nu¨bel U, Strommenger B, Werner G. Emergence and spread of antibiotic-resistant Gram-positive bacterial pathogens. Int J Med Microbiol 2008; 298: 365–377. 2. Deurenberg RH, Stobberingh EE. The evolution of Staphylococcus aureus. Infect Genet Evol 2008; 8: 747–763. 3. Boucillon SK, Johnson BM, Hoban DJ et al. Determining incidence of extended spectrum b-lactamase producing Enterobacteriaceae, vancomycin-resistant Enterococcus faecium and methicillin-resistant Staphylococcus aureus in 38 centres from 17 countries: the PEARLS study 2001–2002. Int J Antimicrob Agents 2004; 24: 119–124. 4. Brink A, Moolman J, da Silva MC, Botha M, the National Antibiotic Surveillance Forum. Antimicrobial susceptibility profile of selected bacteraemic pathogens from private institutions in South Africa. S Afr Med J 2007; 97: 273–279. 5. Aires de Sousa M, de Lencastre H. Bridges from hospitals to the laboratory: genetic portraits of methicillin-resistant Staphylococcus aureus clones. FEMS Immunol Med Microbiol 2004; 40: 101–111. 6. Enright MC, Robinson DA, Randle G, Feil EJ, Grundmann H, Spratt BG. The evolutionary history of methicillin-resistant Staphylococcus aureus (MRSA). Proc Natl Acad Sci USA 2002; 99: 7687–7692. 7. Robinson DA, Enright MC. Evolutionary models of the emergence of methicillin-resistant Staphylococcus aureus. Antimicrob Agents Chemother 2003; 47: 3926–3934. 8. Nu¨bel U, Roumagnac P, Feldkamp M et al. Frequent emergence and limited geographic dispersal of methicillin-resistant Staphylococcus aureus. Proc Natl Acad Sci USA 2008; 105: 14130–14135. 9. Essa ZI, Connolly C, Essack SY. Staphylococcus aureus from public hospitals in KwaZulu-Natal, South Africa – infection detection and strain-typing. South Afr J Epidemiol Infect 2009; 24: 4–7. 10. Shittu A, Nu¨bel U, Udo E, Lin J, Steven SG. Characterization of methicillin-resistant Staphylococcus aureus isolates from hospitals in KwaZulu-Natal (KZN) province, Republic of South Africa. J Med Microbiol 2009; 9 (Pt 9): 1219–1226. 11. Clinical Laboratory Standards Institute. Performance standards for antimicrobial susceptibility testing: seventeenth informational supplement.


792

12.

13.

14.

15.

16.

17.

18.

19.

20.


CLSI document M100-S17. Wayne, PA: Clinical Laboratory Standards Institute, 2007. Clinical Laboratory Standards Institute. Performance standards for antimicrobial susceptibility testing: eighteenth informational supplement. CLSI document M100-S18. Wayne, PA: Clinical Laboratory Standards Institute, 2008. Reed KD, Stemper ME, Shukla SK. Pulsed-field gel electrophoresis of methicillin-resistant Staphylococcus aureus. Methods Mol Biol 2007; 391: 59–69. Murchan S, Kaufmann ME, Deplano A et al. Harmonization of pulsedfield gel electrophoresis protocols for epidemiological typing of strains of methicillin-resistant Staphylococcus aureus: a single approach developed by consensus in 10 European laboratories and its application for tracing the spread of related strains. J Clin Microbiol 2003; 41: 1574–1585. Milheiriço C, Oliveira DC, de Lencastre H. Update to the mupltiplex PCR strategy for assignment of mec element types in Staphylococcus aureus. Antimicrob Agents Chemother 2007; 51: 3374–3377. Erratum in: Antimicrob Agents Chemother 2007; 51: 4537. Kondo Y, Ito T, Ma XX et al. Combination of multiplex PCRs for staphylococcal cassette chromosome mec type assignment: rapid identification system for mec, ccr and major differences in junkyard regions. Antimicrob Agents Chemother 2007; 51: 264–274. Lina G, Pie´mont Y, Godail-Gamot F et al. Involvement of Panton-Valentine Leukocidin–Producing Staphylococcus aureus in primary skin infections and pneumonia. Clin Infect Dis 1999; 29: 1128–1132. Enright MC, Day NPJ, Davies CE, Peacock SJ, Spratt BG. Multilocus sequence typing for characterisation of methicillin-resistant and methicillin-susceptible clones of Staphylococcus aureus. J Clin Microbiol 2000; 38: 1008–1015. Harmsen D, Claus H, Witte W et al. Typing of methicillin-resistant Staphylococcus aureus in a University Hospital setting by using novel software for spa repeat determination and database management. J Clin Microbiol 2003; 41: 5442–5448. Goering RV, Shawar RM, Scangarella NE et al. Molecular epidemiology of methicillin-resistant and methicillin-susceptible Staphylococcus aureus isolates from global clinical trials. J Clin Microbiol 2008; 46: 2842–2847.

CMI

21. Oosthuysen WF, Duse´ AG, Marais E. Molecular characterization of methicillin-resistant Staphylococcus aureus in South Africa. Clin Microbiol Infect 2007; 13 (Suppl. 1): Abstract (P1296). 22. Tavares DA, Sa´-Leaõ R, Miragaia M, de Lencastre H. Large screening of CA-MRSA among Staphylococcus aureus colonizing healthy young children living in two areas (urban and rural) of Portugal. BMC Infect Dis 2010; 10: 110. 23. Schuenck RP, Noue´r SA, de Oliveira Winter C et al. Polyclonal presence of non-multiresistant methicillin-resistant Staphylococcus aureus isolates carrying SCCmec IV in health care-associated infections in a hospital in Rio de Janeiro, Brazil. Diagn Microbiol Infect Dis 2009; 64: 434–441. 24. Peck KR, Baek JY, Song JH, Ko KS. Comparison of genotypes and enterotoxin genes between Staphylococcus aureus isolates from blood and nasal colonizers in a Korean Hospital. J Korean Med Sci 2009; 24: 585–591. 25. Smyth DS, McDougal LK, Gran FW et al. Population structure of a hybrid clonal group of methicillin-resistant Staphylococcus aureus, ST239-MRSA-III. PLoS ONE 2009; 5: e8582. doi: 10.1371/journal.pone.0008582. 26. Johnson AP, Pearson A, Duckworth G. Surveillance and epidemiology of MRSA bacteraemia in the UK. J Antimicrob Chemother 2005; 56: 455–462. 27. Gould SWJ, Rollason J, Hilton AC et al. UK epidemic strains of meticillin-resistant Staphylococcus aureus in clinical samples from Malta. J Med Microbiol 2008; 57: 1394–1398. 28. Ruimy R, Maiga A, Armand-Lefevre L et al. The carriage population of Staphylococcus aureus from Mali is composed of a combination of pandemic clones and the divergent Panton-Valentine Leukocidin-positive genotype ST152. J Bacteriol 2008; 190: 3962–3968. 29. Grundmann H, Aanensen DM, van den Wijngaard CC et al. Geographic distribution of Staphylococcus aureus causing invasive infections in europe: a molecular-epidemiological analysis. PLoS Med 2010; 7: e1000215 doi:10.1371/journal.pmed.1000215. 30. Groome MJ, Albrich W, Khoosal M, Wadula J, Madhi SA. Staphylococcus aureus bacteraemia on admission in paediatric patients at Chris Hani Baragwanath Hosptial, Soweto. South Afr J Epidemiol Infect 2009; 24: 26–27.
