In contrast to methicillin-resistant Staphylococcus aureus, little is known of the distribution of spa types among methicillin-susceptible S. aureus (MSSA). We have analyzed 101 nonrepetitive invasive MSSA isolates from infected patients, consecutively isolated during 14months between 2006 and 2007 at University Hospital Basel. They were genetically characterized according to S. aureus protein A (spa) types and important virulence-associated genes. Sixty-five different spa types corresponding to nine different spa clonal complexes were observed. Analysis of different virulence genes showed a frequency of 17% for toxic-shock syndrome toxin and 5% for exfoliative toxin D. In conclusion, spa typing revealed a great genetic diversity without predominant spa type, not providing evidence for clonal spreadin

Fenner, L.

Frei, R.

Widmer, A.

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BRIEF REPORTMolecular epidemiology of invasivemethicillin-susceptible Staphylococcus aureus strainscirculating at a Swiss University HospitalL. Fenner & A. F. Widmer & R. FreiReceived: 31 July 2007 /Accepted: 14 January 2008 /Published online: 26 February 2008# Springer-Verlag 2008Abstract In contrast to methicillin-resistant Staphylococ-cus aureus, little is known of the distribution of spa typesamong methicillin-susceptible S. aureus (MSSA). We haveanalyzed 101 nonrepetitive invasive MSSA isolates frominfected patients, consecutively isolated during 14 monthsbetween 2006 and 2007 at University Hospital Basel. Theywere genetically characterized according to S. aureusprotein A (spa) types and important virulence-associatedgenes. Sixty-five different spa types corresponding to ninedifferent spa clonal complexes were observed. Analysis ofdifferent virulence genes showed a frequency of 17% fortoxic-shock syndrome toxin and 5% for exfoliative toxin D.In conclusion, spa typing revealed a great genetic diversitywithout predominant spa type, not providing evidence forclonal spreading.ReportStaphylococcus aureus is an important human pathogen andis isolated in up to 25% of nosocomial infections. S. aureusbacteraemia is associated with substantial excess morbidityand mortality worldwide [1]. A recent study from theUnited States showed a predominance of one clonalcomplex (ST8) among methicillin-susceptible S. aureus(MSSA) strains, whereas the remaining strains weregenotypically heterogeneous [2]. Strain typing is anestablished tool for surveillance and determination of strainrelatedness. Sequence-based methods such as protein Agene (spa) strain typing offer the opportunity of reproduc-ible and comparable results and have been successfullyused in epidemiological studies [3, 4]. To date, relativelylittle data exist on spa types and the molecular epidemiol-ogy of invasive MSSA strains in contrast to methicillin-resistant S. aureus (MRSA). In this study, our goal was toassess the frequency of circulating invasive MSSA spatypes isolated from primarily sterile sites in patients atUniversity Hospital Basel (UHBS), Switzerland, and todetect virulence associated genes such as Panton-Valentineleukocidin (PVL), toxic shock syndrome toxin (tst),exfoliative toxin A, B, and D (eta, etb, etd).As standard operating procedure, invasive MSSA iso-lates from primarily sterile sites (cerebro-spinal fluid, sterilebody fluids, biopsies, blood cultures) are routinely saved at−70°C at our laboratory. The isolates were identified as S.aureus on the basis of positive catalase, clumping factor(Slidex StaphPlus, bioMérieux, Marcy l'Etoile, France), andaurease (RAPIDECstaph, bioMérieux). Only one isolate perpatient was included. DNA was extracted with LCMagnaPure system (Roche Diagnostics, Rotkreuz, Switzer-land) according to manufacturer’s instruction. The spa genewas amplified using primers spa-1113f and spa-1514r andsequencing was performed as previously described [4].Sequences were analyzed with Ridom StaphType softwareversion 1.4 (Ridom GmbH, Würzburg, Germany). Cluster-ing analysis into spa clonal complexes (spa-CC) was doneEur J Clin Microbiol Infect Dis (2008) 27:623–626DOI 10.1007/s10096-008-0463-0L. Fenner :R. Frei (*)Microbiology Laboratory, University Hospital Basel,Petersgraben 4,4031 Basel, Switzerlande-mail: rfrei@uhbs.chA. F. WidmerDivision of Infectious Diseases and Hospital Epidemiology,University Hospital Basel,Basel, Switzerlandusing default parameters (spa types are clustered if cost isless than or equal to 6 and spa types shorter than 5 repeatsare excluded). PVL, tst, eta, etb, and etd genes weredetected by PCR as described elsewhere [5–7]. Amplifica-tion of the 16S rRNA gene was used to confirm absence ofPCR inhibitors. PCR products were visualized by electro-phoresis on ethidium bromide-stained 3% agarose gels.Antibiotic susceptibility was tested applying a microbrothdilution procedure (MERLIN Diagnostika, Bornheim-Her-sel, Germany) and interpreted in accordance with theClinical and Laboratory Standards Institute (http://www.clsi.org).A total of 105 patients with MSSA infection during a 14-month period in 2006–2007 were recorded. Isolates of 101patients were analyzed; four isolates were not available.The median age of patients was 61 years (interquartilerange 45–74) with 63.4% being males. The highestproportions of MSSA strains were isolated from thesurgical department (42%), emergency department (31%),internal medicine (11%), and intensive care units (10%).Blood culture was the most common body site; the sameorganism was isolated from another source in 11.2% (9isolates; not included to avoid double counting). Specimensfrom joint, bone, and skin/soft tissue each accounted for5.9% (6 isolates). A total of 65 different spa typescorresponding to nine different spa-CC were observed(Table 1), and 51 strains had a unique spa type. Elevennewly recognized spa types were found which havenever been deposited before in the spa database (http://www.spaserver.ridom.de). The ten most frequent spa typesTable 1 Distribution of 101 invasive methicillin-susceptible S. aureus (MSSA) strains by spa clonal complex (spa-CC), presence of virulence-associated determinants, and antibiotic resistance profileSpa-CC Spa Type Frequency PVL etd tst eta/etb Penicillin R Clarithromycin R Quinolone Rb Site of IsolationBlood Other thanbloodn n n n n n (%) n (%) n (%) n nCC084/085 t084 (8), t085 (1), t091 (1),t094 (1), t254 (1), t279 (1),t346 (2), t803 (1), t2432 (1),t2434 (1)18 1 16 (89) 15 3CC012 t012 (3), t021 (2), t093 (1),t122 (2), t298 (1), t665 (1),t913 (1), t1306 (1), t1836(1),t2437 (1)14 1 8 13 (93) 1 (7.1) 11 3CC002 t002 (7), t062 (2), t548 (2) 11 5 5 (45.5) 3 (27.3) 1 (9) 10 1CC376 t359 (3), t376 (1), t1198 (1),t1897 (2)7 1 2 5 (71.4) 7 0CC078 t056 (4), t078 (1), t258 (1) 6 2 1 (16.7) 4 2CC005 t005 (1), t223 (1), t712 (1),t790 (1), t902 (1), t2439 (1)6 3 (50) 1 (16.7) 4 2CC024 t008 (2), t024 (1), t190 (1) 4 1 (25) 4 0CC888 t127 (1), t160 (1), t888 (1),t2433 (1)4 2 (50) 2 2CC230 t230 (1), t550 (1), t571 (1) 3 1 3 (100) 1 (33.3) 1 2Others a t015 (6), t026 (4), t136 (1),t153 (1), t159 (1), t163 (1),t216 (1), t330 (1), t350 (1),t645 (1), t777 (1), t1662 (1),t1992 (1), t2435 (1), t2438 (1),t2441 (1), t2442 (1), t2443 (1),t2476 (1),28 1 3 1 21 (75) 22 6Total 101 2 5 17 2 70 5 2 80 21Previously published MSSA spa types are in italics [3, 8]. Empty cells denote a negative resulteta/etb exfoliative toxin A/B, etd exfoliative toxin D, PVL Panton-Valentine leukocidin toxin, R resistant, tst toxic shock syndrome toxina Includes strains that were not typeable, singleton, no founder or excluded from analysis (shorter than five repeats)b Resistant to at least one of these antibiotic compounds: levofloxacin, ciprofloxacin, norfloxacin624 Eur J Clin Microbiol Infect Dis (2008) 27:623–626[t084 (8 strains), t002 (7), t015 (6), t026 (4), t056 (4), t012(3), t359 (3), t008 (2), t021 (2), t062 (2)] accounted for40.6% of all isolates. Comparing our MSSA spa types withthose deposited in the spa server for MRSA and MSSAstrains, t002 is the fourth most frequent spa type worldwideamong MSSA and MRSA isolates. t008, t015, t024, andt084 correspond to the top ten spa types in this largedatabase (http://www.spaserver.ridom.de). Some of our spatypes are similar to those previously reported in MSSAstrains (Table 1, spa types in italics) [3, 8]. Geneticdiversity of our study isolates exceeded that of a previouslypublished study where 14 different spa types among 84MSSA strains were detected [3]. A recent study fromFrance on MSSA bloodstream infections using pulsed-fieldgel electrophoresis (PFGE) for genetic analysis showed asmall number of PFGE divisions among isolates and wereassociated with epidemic phenomena in healthcare institu-tions [9]. If spa typing or spa-CC data were analyzed by thetype of ward, no cluster with the same MSSA spa type atthe same time was observed. The findings of moleculartyping were supported by epidemiological data fromsurveillance: there was no evidence for an outbreaksituation during the study period.S. aureus produces a variety of extracellular toxins.They are associated with abscesses, furunculosis, andnecroticing pneumonia or toxic shock syndrome [5, 6].The tst gene was detected in 17% of cases, etd in 5%, PVLand eta/etb each in 2%. One isolate carried both PVL andetd (spa type t1198). Every etd-positive strain wasassociated with a different spa type, 17 tst-positive strainswith 13 different types (4 strains of spa t002). In ourstudy, the percentage of tst gene-positive invasive MSSAstrains was comparable with MSSA strains from blood-stream infections [9] but lower than in a collection ofMSSA strains mainly from sites other than blood [3]. Alltoxin positive strains were isolated from blood culturesexcept for two tst-positive strains (from soft tissue and ajoint biopsy).In contrast to recent studies from France and Germany,the antibiotic resistance rates (Table 1) of quinolones andmacrolides were low [3, 9]. However, 93% and 89% of spa-CC012 and spa-CC084/CC085 were penicillin-resistant,respectively. Only 5% of all strains were macrolideresistant, not allowing meaningful interpretation. Theprevalence of methicillin-resistant S. aureus (MRSA) inour institution is low [10]; only two invasive isolates of S.aureus were found to be methicillin resistant and weretherefore excluded.Nine of 65 spa types representing 26.7% of MSSAstrains were also represented in our local MRSA spadatabase (t002, t008, t012, t015, t024, t026, t163, t230,t548). This genetic similarity between MRSA and MSSA isconsistent with other reports [3, 8, 11]. However, these spatypes are also frequent types worldwide and thereforeimportation of MRSA strains seems more likely thanacquisition of methicillin-resistance genes among the localMSSA strain pool as suggested previously [8, 11]. But thecommon genetic background among MSSA and MRSAstrains indicates a certain selective advantage regardless ofresistance and accessory virulence gene profile.In conclusion, spa typing revealed a great geneticdiversity among invasive MSSA strains consecutivelyisolated from primarily sterile sites without a predominantspa type or spa-CC. Besides worldwide common spa typesand spa-CC, many rare and newly recognized spa typeswere observed. There was no evidence for MSSA cross-infections among patients. tst was the most frequentvirulence gene detected among MSSA isolates. Furtherspa typing data on MSSA strains from other places wouldallow a more comprehensive understanding of the geneticbackground.Acknowledgements This study was supported by the scientificresearch fund of the University Hospital Basel.References1. Kaech C, Elzi L, Sendi P, Frei R, Laifer G, Bassetti S, Fluckiger U(2006) Course and outcome of Staphylococcus aureus bacterae-mia: a retrospective analysis of 308 episodes in a Swiss tertiary-care centre. Clin Microbiol Infect 12:345–3522. Miller LG, Perdreau-Remington F, Bayer AS, Diep B, Tan N,Bharadwa K, Tsui J, Perlroth J, Shay A, Tagudar G, Ibebuogu U,Spellberg B (2007) Clinical and epidemiologic characteristicscannot distinguish community-associated methicillin-resistantStaphylococcus aureus infection from methicillin-susceptible S.aureus infection: a prospective investigation. Clin Infect Dis44:471–4823. Layer F, Ghebremedhin B, Konig W, Konig B (2006) Heteroge-neity of methicillin-susceptible Staphylococcus aureus strains at aGerman University Hospital implicates the circulating-strain poolas a potential source of emerging methicillin-resistant S. aureusclones. J Clin Microbiol 44:2179–21854. Strommenger B, Kettlitz C, Weniger T, Harmsen D, Friedrich AW,Witte W (2006) Assignment of Staphylococcus isolates to groupsby spa typing, SmaI macrorestriction analysis, and multilocussequence typing. J Clin Microbiol 44:2533–25405. Lina G, Piemont Y, Godail-Gamot F, Bes M, Peter MO,Gauduchon V, Vandenesch F, Etienne J (1999) Involvement ofPanton-Valentine leukocidin-producing Staphylococcus aureus inprimary skin infections and pneumonia. Clin Infect Dis 29:1128–11326. Yamasaki O, Tristan A, Yamaguchi T, Sugai M, Lina G, Bes M,Vandenesch F, Etienne J (2006) Distribution of the exfoliativetoxin D gene in clinical Staphylococcus aureus isolates in France.Clin Microbiol Infect 12:585–5887. Mehrotra M, Wang G, Johnson WM (2000) Multiplex PCR fordetection of genes for Staphylococcus aureus enterotoxins,Eur J Clin Microbiol Infect Dis (2008) 27:623–626 625exfoliative toxins, toxic shock syndrome toxin 1, and methicillinresistance. J Clin Microbiol 38:1032–10358. Hallin M, Denis O, Deplano A, De Mendonca R, De Ryck R,Rottiers S, Struelens MJ (2007) Genetic relatedness betweenmethicillin-susceptible and methicillin-resistant Staphylococcusaureus: results of a national survey. J Antimicrob Chemother59:465–4729. van der Mee-Marquet N, Epinette C, Loyau J, Arnault L,Domelier AS, Losfelt B, Girard N, Quentin R (2007) Staphylo-coccus aureus strains isolated from bloodstream infectionschanged significantly in 2006. J Clin Microbiol 45:851–85710. Mertz D, Frei R, Jaussi B, Tietz A, Stebler C, Fluckiger U,Widmer AF (2007) Throat swabs are necessary to reliably detectcarriers of Staphylococcus aureus. Clin Infect Dis 45:475–47711. Aires de Sousa M, Conceicao T, Simas C, de Lencastre H (2005)Comparison of genetic backgrounds of methicillin-resistant and -susceptible Staphylococcus aureus isolates from Portuguesehospitals and the community. J Clin Microbiol 43:5150–5157626 Eur J Clin Microbiol Infect Dis (2008) 27:623–626

Molecular epidemiology of invasive methicillin-susceptible Staphylococcus aureus strains circulating at a Swiss University Hospital

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Molecular epidemiology of invasive methicillin-susceptible Staphylococcus aureus strains circulating at a Swiss University Hospital

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