Rhesus Macaques (Macaca mulatta) Are Natural Hosts of Specific Staphylococcus aureus Lineages

Rhesus Macaques (Macaca mulatta) Are Natural Hosts of Specific Staphylococcus aureus Lineages Sanne van den Berg1*, Willem J. B. van Wamel1, Susan V. Snijders1, Boudewijn Ouwerling2, Corné P. de Vogel1, Hélène A. Boelens1, Rob J. L. Willems3, Xander W. Huijsdens4, Frank A. W. Verreck5, Ivanela Kondova2, Peter J. Heidt2, Henri A. Verbrugh1, Alex van Belkum1 1 Department of Medical Microbiology and Infectious Diseases, Erasmus MC, Rotterdam, The Netherlands, 2 Animal Science Department, Biomedical Primate Research Centre, Rijswijk, The Netherlands, 3 Department of Medical Microbiology, University Medical Centre Utrecht, Utrecht, The Netherlands, 4 Laboratory for Infectious Diseases and Screening, National Institute for Public Health and the Environment, Bilthoven, The Netherlands, 5 Department of Parasitology, Biomedical Primate Research Centre, Rijswijk, The Netherlands Abstract Currently, there is no animal model known that mimics natural nasal colonization by Staphylococcus aureus in humans. We investigated whether rhesus macaques are natural nasal carriers of S. aureus. Nasal swabs were taken from 731 macaques. S. aureus isolates were typed by pulsed-field gel electrophoresis (PFGE), spa repeat sequencing and multi-locus sequence typing (MLST), and compared with human strains. Furthermore, the isolates were characterized by several PCRs. Thirty-nine percent of 731 macaques were positive for S. aureus. In general, the macaque S. aureus isolates differed from human strains as they formed separate PFGE clusters, 50% of the isolates were untypeable by agr genotyping, 17 new spa types were identified, which all belonged to new sequence types (STs). Furthermore, 66% of macaque isolates were negative for all superantigen genes. To determine S. aureus nasal colonization, three nasal swabs from 48 duo-housed macaques were taken during a 5 month period. In addition, sera were analyzed for immunoglobulin G and A levels directed against 40 staphylococcal proteins using a bead-based flow cytometry technique. Nineteen percent of the animals were negative for S. aureus, and 17% were three times positive. S. aureus strains were easily exchanged between macaques. The antibody response was less pronounced in macaques compared to humans, and nasal carrier status was not associated with differences in serum anti-staphylococcal antibody levels. In conclusion, rhesus macaques are natural hosts of S. aureus, carrying host-specific lineages. Our data indicate that rhesus macaques are useful as an autologous model for studying S. aureus nasal colonization and infection prevention. Citation: van den Berg S, van Wamel WJB, Snijders SV, Ouwerling B, de Vogel CP, et al. (2011) Rhesus Macaques (Macaca mulatta) Are Natural Hosts of Specific Staphylococcus aureus Lineages. PLoS ONE 6(10): e26170. doi:10.1371/journal.pone.0026170 Editor: Vance G. Fowler, Duke University Medical Center, United States of America Received March 11, 2011; Accepted September 21, 2011; Published October 20, 2011 Copyright: ß 2011 van den Berg 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: S.v.d.B. and A.v.B. were in part supported financially by the Top Institute Pharma project T4-213. B.O. and I.K. were in part supported financially by EUPRIM-NET2, grant agreement no. 262443. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. No additional external funding received for this study. Competing Interests: The authors have read the journals’ policy and have the following conflicts: S.v.d.B. and A.v.B. were in part supported by the Top Institute Pharma project T4-213. This does not alter the authors’ adherence to all the PLoS ONE policies on sharing data and materials. * E-mail: [email protected] Introduction S. aureus [7,8]. Humans can acquire these S. aureus strains during intensive short-term exposure to livestock, but in most cases the In the light of the rapid, worldwide emergence of antibiotic strain is lost again within 24 hours [9]. However, lack of a natural, resistance in and the lack of an effective long-term elimination human-like animal model of nasal S. aureus carriage is still a strategy against Staphylococcus aureus (S. aureus) nasal carriage, new problem. Therefore, we investigated whether a non-human approaches are needed to prevent staphylococcal carriage and its primate could provide a natural model for human nasal carriage consequent diseases. Many different S. aureus animal models have of S. aureus. been described for studying the pathogenesis of S. aureus The rhesus macaque (Macaca mulatta) belongs to the old world colonization and infection. These models have provided insight monkeys and has been used in several studies involving S. aureus. into the role of bacterial virulence genes and have assisted in the Kuklin et al. used rhesus macaques to study the immunogenicity of estimation of vaccine efficacy. Models have been set up in various IsdB [10]. Protection against lethal SEB aerosol exposure by species, such as insects, worms, mice, rats, guinea pigs, hamsters, passive transfer of SEB-specific antibodies was also studied in chickens, rabbits, sheep, dogs, pigs, and cows [1–4]. Notably, most macaques [11]. In addition to these protection studies, rhesus of these animals, unlike humans, are not natural nasal carriers of S. macaques were also used for safety evaluations. For example, the aureus, only pigs, sheep, and cows may be naturally colonized by S. tolerability and potential toxicity of the thrombolytic agent aureus. For instance, S. aureus sequence type (ST) 398 strains belong staphylokinase was investigated in healthy rhesus macaques [12]. to a biotype associated with pigs and other species of livestock To our knowledge, natural nasal S. aureus carriage and the [5,6]. S. aureus strain RF122 is a member of a bovine mastitis- consequences on natural immunity in rhesus macaques has never associated clone that is genetically different from human clones of been studied before. PLoS ONE | www.plosone.org 1 October 2011 | Volume 6 | Issue 10 | e26170  Rhesus Macaques as Natural Hosts of S. aureus Figure 1. Dendrogram of the PFGE data of S. aureus strains isolated from rhesus macaques and humans. Blue color indicates S. aureus strains isolated from rhesus macaques, red color indicates S. aureus strains isolated from humans. Five clusters consisted of only rhesus macaque isolates, one comprised only human isolates, and five clusters included isolates from both macaques and humans. doi:10.1371/journal.pone.0026170.g001 Using a cross sectional setup, we isolated 287 S. aureus strains Indian, Burmese and Chinese origin. These macaques were from 731 rhesus macaques after nasal sampling. We compared S. housed in groups of 2–44 individuals. Furthermore, 48 young aureus strains isolated from rhesus macaques and humans. rhesus macaques that were recently imported from China were Furthermore, we followed a group of 48 rhesus macaques in time followed in time for studying the persistence of S. aureus nasal for studying persistence of nasal carriage of S. aureus. In addition, carriage as well as their serum anti-staphylococcal antibody levels. serum samples from these macaques were analyzed for anti- These animals were duo-housed in 4 different animal rooms. staphylococcal immunoglobulin G and A (IgG and IgA) levels. Physical contact with the macaques in the neighbouring cage was possible. In each room 2 groups of cages were located opposite to Materials and Methods each other. Study population Human S. aureus strains For comparison of S. aureus strains isolated from rhesus For reasons of comparison, 56 human isolates of S. aureus were macaques with those from humans, 731 rhesus macaques from included. These carriage (n = 30) and bacteremia derived (n = 20) the breeding colony of the Biomedical Primate Research Centre MSSA isolates have been described before [13,14]. Three MSSA (Rijswijk, The Netherlands) were studied. These animals were of isolates from animal care-takers and 3 S. aureus strains for which PLoS ONE | www.plosone.org 2 October 2011 | Volume 6 | Issue 10 | e26170  Rhesus Macaques as Natural Hosts of S. aureus Table 1. Different spa types found in 108 representative S. For bacteremia derived isolates, S. aureus strains were collected for aureus strains isolated from rhesus macaques. routine culture. The Medical Ethics Committee of the Erasmus Medical Centre Rotterdam approved the study (MEC-2007-106, addendum 2). spa type No. of isolates Percentage of isolates t189 1 0.9 Sampling procedures A total of 731 macaques were sampled once for nasal carriage of t516 21 19.4 S. aureus, while another 48 macaques were screened three times t729 15 13.9 during a 5 month period. Nasal cultures were taken by streaking t786 4 3.7 both anterior nares using a sterile cotton swab (Swab Transystem, t1814 3 2.8 Greiner Bio One, Alphen aan de Rijn, The Netherlands) during t4167 new 11 10.2 regular animal medical check-up. All swabs were processed within 24 hours. Nasal swabs were plated on a Columbia sheep blood t4168 new 18 16.7 agar plate-medium (bioTRADING, Mijdrecht, The Netherlands). t4980 new 2 1.9 Plates were read after one and two days of incubation at 35uC. t4981 new 1 0.9 Identification of S. aureus was based on colony morphology and t5010 new 2 1.9 coagulase plasma test (Becton Dickinson, Breda, The Netherlands) t5011 new 1 0.9 and confirmed by API Staph (bioMérieux, Boxtel, The Nether- t5012 new 1 0.9 lands). t5013 new 6 5.6 Pulsed-field gel electrophoresis t5014 new 1 0.9 Pulsed-field gel electrophoresis (PFGE) of SmaI digested t5015 new 4 3.7 chromosomal DNA from all S. aureus strains from rhesus macaques t5016 new 1 0.9 and 56 strains from humans was performed as described t5017 new 5 4.6 previously [15]. Relatedness among the PFGE profiles was t5018 new 5 4.6 evaluated with Bionumerics software (version 3.0; Applied Maths, t5135 new 3 2.8 Ghent, Belgium). A dendrogram was produced using the Dice coefficient and an unweighted-pair group method using arithmetic t5136 new 1 0.9 averages (UPGMA). Band tolerance was set at 2.0%. t5137 new 1 0.9 t5458 new 1 0.9 Anti-staphylococcal antibodies doi:10.1371/journal.pone.0026170.t001 IgG and IgA antibody levels in serum directed against the following antigens were semi-quantified: S. aureus proteins clumping factor A and B (ClfA and ClfB); surface protein G the genome sequence is known were included as well (N315, (SasG); iron-responsive surface determinants A and H (IsdA and Mu50, MRSA252). IsdH); fibronectin-binding proteins A and B (FnbpA and FnbpB); serine-aspartate dipeptide repeat protein D and E (SdrD and Ethics statement SdrE); staphylococcal enterotoxins A-E, G-J, M-O, Q, and R Sampling of the longitudinally screened macaques was ap- (SEA - SEE, SEG - SEJ, SEM - SEO, SEQ, SER); toxic shock proved by the Animal Experiments committee of the Biomedical syndrome toxin 1 (TSST-1); chemotaxis inhibitory protein of S. Primate Research Centre (Dierexperimentencommissie (DEC), aureus (CHIPS); staphylococcal complement inhibitor (SCIN); which is the ethical committee installed and officially recognised as extracellular fibrinogen-binding protein (Efb); exfoliative toxin A required by the Dutch Law on Experimental Animals and which is and B (ETA and ETB); alpha toxin; c hemolysin B (HlgB); the Dutch analogue for the IACUC). The approval number is: leukocidin (Luk) S-PV, LukF-PV, LukD-PV, and LukE-PV; and DEC#579, dated October 28, 2008. The study was conducted in staphylococcal superantigen-like proteins 1, 3, 5, 9, and 11 (SSL1, compliance with all relevant Dutch laws and in agreement with SSL3, SSL5, SSL9, and SSL11). Antibodies were semi-quantified international and scientific standards and guidelines. simultaneously in a single multiplex assay using a bead-based flow Sampling for bacteriology of the animals in the breeding groups cytometry technique (xMap; Luminex Corporation). Methods was performed as part of their yearly routine health screening, have been described elsewhere [14,16,17], with the exception that which is by Dutch law not considered to be an animal experiment. a 1:50 dilution of R-phycoerythrin (RPE)-conjugated AffiniPure Therefore no permission of the ethical committee was necessary goat anti-human secundairy IgA was used. Tests were performed for this part of the study. All samplings were performed under in independent duplicates, and the median fluorescence intensity ketamine anaesthesia (which is routine for all health checks of non- (MFI) values, reflecting semiquantitative antibody levels, were human primates) and all efforts were made to minimize stress and averaged. In each experiment, control beads (no protein coupled) suffering of the animals. The housing, care and handling of all were included to determine nonspecific binding. In the event of animals were according to the Dutch Law on Experimental nonspecific binding, the nonspecific MFI values were subtracted Animals and the European Directive 86/609/EEC. The research from the antigen-specific results. described is in accordance to the Weatherall Report recommen- Serum samples from 47 out of the 48 rhesus macaques from dations for good welfare. China were analyzed. Anti-staphylococcal antibody levels in these Human samples were described before [13,14]. For carriage sera were compared to those in sera from 20 Dutch healthy isolates and serum samples, volunteers provided written informed human volunteers [14]. Pooled serum from all rhesus macaques consent and the local Medical Ethics Committee of the Erasmus included in the present study or from humans was used as a Medical Centre Rotterdam approved the study (MEC-2007-106). standard. PLoS ONE | www.plosone.org 3 October 2011 | Volume 6 | Issue 10 | e26170  Rhesus Macaques as Natural Hosts of S. aureus Table 2. Relatedness of spa types within each PFGE cluster. spa types PFGE in PFGE cluster cluster Repeat succession 1 - 2 t4980 4 - 16 - 21 - 12 - 12 - 17 - 13 - 39 - 13 t5135 210 - 23 - 34 - 34 - 16 - 17 - 34 - 17 - 17 - 23 - 17 3 - 4 t516 8 - 16 - 2 - 25 - 51 - 68 - 2 - 24 - 2 - 24 t5014 15 - 12 - 16 - 16 - 2 - 25 - 34 - 24 - 17 - 24 - 17 t5015 15 - 12 - 16 - 16 - 25 - 25 - 34 - 24 - 17 - 17 - 17 t5017 8 - 34 - 2 - 43 - 34 - 43 - 43 - 16 - 2 - 17 - 83 5 t516 8 - 16 - 2 - 25 - 51 - 68 - 2 - 24 - 2 - 24 t5011 8 - 16 - 16 - 25 - 25 - 34 - 24 - 17 - 17 - 17 t5015 15 - 12 - 16 - 16 - 25 - 25 - 34 - 24 - 17 - 17 - 17 6 t516 8 - 16 - 2 - 25 - 51 - 68 - 2 - 24 - 2 - 24 t729 7 - 12 - 21 - 17 - 13 - 34 - 34 - 34 - 33 - 34 t786 7 - 12 - 21 - 17 - 13 - 34 - 34 - 33 - 34 t1814 7 - 12 - 21 - 17 - 34 - 34 - 34 - 33 - 34 t5012 8 - 34 - 2 - 43 - 34 - 43 - 43 - 16 - 2 - 17 - 16 t5136 4 - 2 - 17 - 17 - 34 - 24 - 17 - 17 - 17 t5458 210 - 23 - 34 - 17 - 34 - 22 - 34 - 17 - 23 - 34 7 t4167 7 - 23 - 17 - 22 - 249 - 12 - 117 - 24 - 25 - 17 t4168 8 - 23 - 21 - 17 - 13 - 13 - 13 - 24 - 23 t4980 4 - 16 - 21 - 12 - 12 - 17 - 13 - 39 - 13 t5013 7 - 23 - 23 - 17 - 34 - 21 - 12 - 20 - 24 t5016 8 - 34 - 2 - 43 - 34 - 43 - 17 - 83 t5017 8 - 34 - 2 - 43 - 34 - 43 - 43 - 16 - 2 - 17 - 83 t4981 8 - 16 - 2 - 43 - 17 - 34 t5018 8 - 16 - 2 - 43 - 17 - 173 - 34 - 17 - 34 8 t4168 8 - 23 - 21 - 17 - 13 - 13 - 13 - 24 - 23 t5010 8 - 23 - 12 - 17 - 13 - 13 - 13 - 24 - 23 9 t4167 7 - 23 - 17 - 22 - 249 - 12 - 117 - 24 - 25 - 17 t4168 8 - 23 - 21 - 17 - 13 - 13 - 13 - 24 - 23 t5015 15 - 12 - 16 - 16 - 25 - 25 - 34 - 24 - 17 - 17 - 17 t5137 4 - 16 - 13 10 t4168 8 - 23 - 21 - 17 - 13 - 13 - 13 - 24 - 23 11 t189 7 - 23 - 12 - 21 - 17 - 34 doi:10.1371/journal.pone.0026170.t002 DNA isolation amplification primers from a commercial supplier (SeqLab, DNA isolation of S. aureus was performed for representative Goettingen, Germany). The forward and reverse sequence isolates from each PFGE cluster. Total DNA of S. aureus was chromatograms were analyzed with the Ridom StaphType isolated with the QIAamp DNA Mini Kit (QIAGEN, Venlo, The software (Ridom GmbH, Würzburg, Germany). Netherlands) according to the manufacturer’s instructions, or with MagNA Pure LC DNA isolation kit III (bacteria, fungi) using the Multi-locus sequence typing MagNA Pure LC instrument (Roche Diagnostics, Almere, The For one representative S. aureus isolate of each spa type, the Netherlands) [18]. sequence type (ST) was determined using multi-locus sequence typing (MLST) [20]. goeBURST [21], as implement in Phyloviz spa genotyping software (http://www.phyloviz.net/wiki/), and which used the PCR for amplification of the S. aureus protein A (spa) repeat same priority rules for linking STs as eBURST but with a global region was performed according to the published protocol [19]. optimization, was used to infer relatedness between STs. PCR products were purified with the QIAquick PCR Purification goeBURST was run on the whole S. aureus MLST database Kit (QIAGEN, Venlo, The Netherlands) and sequenced using two (http://saureus.mlst.net/; interrogated July 2011) supplemented PLoS ONE | www.plosone.org 4 October 2011 | Volume 6 | Issue 10 | e26170  Rhesus Macaques as Natural Hosts of S. aureus Figure 2. Partial population snapshot of S. aureus. This snapshot was created by goeBURST v1.2 software using the dataset downloaded from http://saureus.mlst.net/ that included 2010 STs representing 3887 isolates. A subset of this dataset (1304 STs, representing 2875 isolates) is presentend in this figure including all major Clonal Complexes (CCs) supplemented with all rhesus macaque ST as far as they were not part of the major CCs. The area of each circle in the goeBURST diagram corresponds to the relative abundance of the STs in the input data. The names of major CCs have been indicated. ST colors refer to the source S. aureus was isolated from. STs representing macaque isolates are indicated in blue and by their ST number; brown colors correspond with community-acquired human isolates; red colors correspond with hospital-acquired human isolates; green colors correspond with animal isolates; yellow colors correspond with S. aureus isolates from food; grey, light green, and light blue colors correspond with isolates with unknown source. doi:10.1371/journal.pone.0026170.g002 with STs of rhesus macaque isolates not present in the S. aureus Results MLST database at the time of interrogation. Clonal complexes (CCs) are defined as STs that are linked through single locus Characterization of S. aureus isolates in rhesus macaques variants (SLVs) and are named on the basis of the predicted A total of 731 macaques was screened once for presence of S. founder ST, which is the ST having the most SLVs. aureus in their nasal cavity. Thirty-nine percent (287/731) of these cultures were positive for S. aureus, clearly indicating that rhesus macaques can be S. aureus nasal carriers. Staphylococcal antigen PCRs S. aureus isolates from the 287 culture positive macaques as well Methicillin resistance was detected with mecA-specific primers as all collected isolates from the 48 macaques that were sampled [22]. Six sets of multiplex PCRs were established to amplify 19 three times (a total of 355 isolates) were typed using molecular superantigen genes (sea to see, seg to ser, seu, tst), eta, etd and agr-1 to - methods including PFGE. This revealed 11 major clusters 4, as described previously [23]. The presence of the scn, chp, clfA, (Figure 1). Five of these consisted of only rhesus macaque isolates, clfB, efb, fnbA, fnbB and tst genes was determined by PCR as well one comprised only human isolates, while five clusters included [13,24–26]. isolates from both macaques and humans. For further characterization of the S. aureus isolates from rhesus Statistical analysis macaques, spa genotyping was performed on representative Statistical analyses were performed with SPSS software, version isolates from each PFGE cluster (n = 108) (Table 1). These isolates 15.0 (SPSS). The Mann-Whitney U test was used to compare were all spa-positive, re-confirming that these were indeed S. aureus median differences in anti-staphylococcal antibody levels. Differ- strains. This revealed 22 different spa types, of which five have ences were considered statistically significant when 2-sided P- already been described before. Consequently, we identified 17 new values were ,0.05. spa types not included in the Ridom SpaServer database at the PLoS ONE | www.plosone.org 5 October 2011 | Volume 6 | Issue 10 | e26170  Rhesus Macaques as Natural Hosts of S. aureus Table 3. Presence of genes encoding superantigens, exfoliative toxins, and agr types in 32 representative S. aureus strains isolated from rhesus macaques. Genea Gene PFGE Origin isolates Isolate no. seg sei sem sen seo etd agr-1 agr-2 agr-3 agr-4 profile cluster in PFGE clusterb 08-1178 2 2 2 2 2 2 + 2 2 2 1 5 RM 08-1192 2 2 2 2 2 2 + 2 2 2 1 4 RM 08-1276 2 2 2 2 2 2 + 2 2 2 1 7 RM 08-1494 2 2 2 2 2 2 + 2 2 2 1 7 RM 08-1495 2 2 2 2 2 2 + 2 2 2 1 7 RM 08-1185 + + + + + 2 + 2 2 2 2 8 RM 08-1415 + + + + + 2 + 2 2 2 2 8 RM 08-1197 + 2 2 2 2 2 2 2 2 + 3 6 RM+HV 08-1255 + 2 2 2 2 2 2 2 2 2 4 11 RM+HV 08-1299 2 2 2 2 2 2 2 2 2 + 5 5 RM 08-1514 2 2 2 2 2 2 2 2 2 + 5 5 RM 08-1332 2 2 2 2 2 2 2 + 2 2 6 4 RM 08-1342 2 2 2 2 2 2 2 2 + 2 7 6 RM+HV 08-1404 2 2 2 2 2 2 2 2 2 2 8 5 RM 08-1412 2 2 2 2 2 2 2 2 2 2 8 6 RM+HV 08-1423 2 2 2 2 2 2 2 2 2 2 8 7 RM 08-1430 2 2 2 2 2 2 2 2 2 2 8 7 RM 08-1432 2 2 2 2 2 2 2 2 2 2 8 7 RM 08-1487 2 2 2 2 2 2 2 2 2 2 8 6 RM+HV 08-1504 2 2 2 2 2 2 2 2 2 2 8 7 RM 08-1507 2 2 2 2 2 2 2 2 2 2 8 7 RM 08-1528 2 2 2 2 2 2 2 2 2 2 8 7 RM 08-1737 2 2 2 2 2 2 2 2 2 2 8 2 RM+HV 08-1749 2 2 2 2 2 2 2 2 2 2 8 7 RM 08-1756 2 2 2 2 2 2 2 2 2 2 8 7 RM 08-1419 2 2 2 2 2 + 2 2 + 2 9 7 RM 08-1420 2 2 2 2 2 + 2 2 + 2 9 7 RM 08-1486 + 2 + + + 2 2 2 2 2 10 7 RM 08-1583 + + + + + 2 2 2 2 2 11 6 RM+HV 08-1602 + + + + + 2 2 2 + 2 12 9 RM+HV 08-1765 + + + + + 2 2 2 + 2 12 7 RM 08-1630 2 2 2 2 2 + 2 2 2 2 13 2 RM+HV No. of positive 8 (25) 5 (16) 6 (19) 6 (19) 6 (19) 3 (9) 7 (22) 1 (3) 5 (16) 3 (9) isolates (%) a All isolates were negative for sea, seb, sec, sed, see, seh, sej, sek, sel, sep, seq, ser, seu, tst, and eta. b Isolates belong to typically rhesus macaque (RM) PFGE clusters or to mixed clusters including both macaque and healthy human volunteer (HV) strains. doi:10.1371/journal.pone.0026170.t003 time of analysis. These new spa types comprised 59% of the present in the S. aureus MLST database at the time of isolates. The most prominent clone was t516, which comprised 21 interrogation. ST2099, ST2100 and ST2134 were part of isolates (19%). Spa types t4168, t729 and t4167 were represented CC45, which mostly contained STs found in human S. aureus by 18 (17%), 15 (14%) and 11 (10%) isolates, respectively. Nine spa isolates. ST2099 and ST2100 were SLVs. Isolates with these 3 STs types were present as single isolates. Each major PFGE cluster were found in different PFGE clusters. ST2094 had relations with comprised 1 to 8 different spa types, though the repeat succession STs in CC1, which contained a lot of STs found in human isolates of different spa types within a cluster were clearly related in some as well. The PFGE pattern of this S. aureus isolate differed from all cases (Table 2). other macaque isolates, as this isolate was found in PFGE cluster For one S. aureus isolate of each spa type, we performed MLST 11, in which this isolate was the only one from macaque origin. All and compared STs found in macaque strains with all STs available other STs found in the macaque isolates (ST1760, ST1761, in the S. aureus MLST database at the moment of interrogation ST1768, ST2095, ST2096, ST2097, ST2098, ST2105, ST2106, (Figure 2). Seventeen different STs were found, which were all not ST2107, ST2108, ST2119, ST2120) were not part of the larger PLoS ONE | www.plosone.org 6 October 2011 | Volume 6 | Issue 10 | e26170  Rhesus Macaques as Natural Hosts of S. aureus PLoS ONE | www.plosone.org 7 October 2011 | Volume 6 | Issue 10 | e26170  Rhesus Macaques as Natural Hosts of S. aureus Figure 3. Persistence of S. aureus nasal carriage in rhesus macaques. Macaques were duo-housed. Physical contact to the macaques in the neighbouring cage was possible. Each block indicates an animal room, horizontal lines in blocks indicate which macaques were not able to have physical contact. Red color indicates the ‘‘epidemic’’ strain in each room. – indicates a S. aureus negative nasal culture. * indicates that the isolate was not spa genotyped itself, spa type was derived from typed isolates with identical PFGE pattern. 1 indicates that the isolate was not spa genotyped, and this could not be derived from typed isolates with identical PFGE pattern. S. aureus strains were frequently exchanged between macaques in the same room. doi:10.1371/journal.pone.0026170.g003 CCs, but were present as singletons. ST1760 and ST2096 as well PFGE type F, spa type t4167). In some cases, a macaque did not as ST1768 and ST2095 were SLVs, and were both found in PFGE acquire the epidemic strain, e.g. the macaque in animal room 2, clusters 7 and 6, respectively, clusters with mainly isolates from cage 6 had two nasal cultures positive with a S. aureus isolate with macaques. Isolates with STs present as a singleton were distributed PFGE type C9, while the epidemic isolate had PFGE type D. among PFGE clusters 4 to 9, which were clusters with mainly isolates from rhesus macaques. Anti-staphylococcal antibodies in rhesus macaques Next, we determined the genomic presence of mecA, 19 Serum samples from 47 of the 48 macaques that were screened superantigen genes, and eta and etd by PCR. None of the 108 thee times for nasal carriage were analyzed for anti-staphylococcal selected isolates harboured the mecA gene, which indicates that antibodies directed against 40 different S. aureus proteins. The MFI none of these isolates were MRSA. Thirty-two isolates that were values reflecting the serum IgG and IgA levels for each macaque representative for each spa type were selected for testing presence and each antibody isotype are shown in Figure 4. An extensive of superantigen genes and eta and etd (Table 3). These genes were diversity in individual antibody responses was observed, similar to absent in 66% (21/32) of these isolates. The etd gene was detected the findings in humans. Individual macaques had high antibody in 9% of the isolates (3/32), 16% of the isolates were sei-positive levels against a number of antigens, while anti-staphylococcal (5/32), 19% were positive for sem, sen, and seo (6/32), and 25% antibody levels against other antigens were low. were positive for seg (8/32). Genes encoding SEA to SEE, SEH, For most antigens, there was no apparent quantitative SEJ to SEL, SEP to SER, SEU, TSST-1 and ETA were absent in difference in antibody level between macaques with 0, 1, 2, or 3 all 32 isolates. Only 6 isolates harboured a complete or incomplete positive cultures. However, the median serum levels of IgG egc locus. Four of these were strains from PFGE clusters comprising directed against ClfA were significantly higher in rhesus macaques only macaque isolates, while 2 isolates belong to clusters including with one positive culture than in those without positive culture both macaque and human isolates. (MFI value, 657 vs. 191; P,0.05). Additionally, median IgA levels These 32 isolates were also agr genotyped (Table 3). The most were higher in macaques without a positive culture than in those common agr type was type I (22%), followed by agr type III (16%), with three positive cultures for LukF (532 vs. 167; P,0.05). In type IV (9%), and type II (3%). Sixteen isolates (50%) did not macaques with three positive cultures, median IgA levels were belong to any of the 4 known agr types, which is intriguing. Isolates higher than in macaques without positive culture for SdrE (881 vs. with agr type I or type II belonged to PFGE clusters comprising 200; P,0.05). In macaques with one positive culture, median IgA only macaque isolates, while isolates with other agr types belonged levels were higher than in macaques with three positive cultures to clusters including both macaque and human isolates. Overall, for LukD (1580 vs. 599; P,0.005), SEC (658 vs. 23; P,0.05), and 13 combinations of agr type and toxin genes were found. Two of SSL11 (204 vs 22; P,0.05). In general, a larger number of positive the agr type I isolates, two of the agr type III isolates and two of the cultures was not related to elevated or decreased anti-staphylo- unknown agr type isolates harboured a complete or incomplete egc coccal antibody levels. Other comparisons were also made (non- locus. Five agr type I isolates, the single agr type II isolate, one agr carriers vs. intermittent vs. persistent carriers; carriers of the most type III isolate, two agr type IV isolates, and 12 isolates with an prominent isolate in the animal room vs. carriers of another unknown agr type harboured none of the superantigen and isolate). Again, no association was found between nasal carrier exfoliative toxin genes. S. aureus isolates with the same agr type and status and anti-staphylococcal antibody levels in serum (data not toxin genes were distributed among the different PFGE clusters. shown). Isolates from rhesus macaques differ from those from humans in S. aureus nasal carriage in rhesus macaques PFGE type, spa type, agr type, and prevalence of superantigen Forty-eight (4 groups of 12) macaques were screened three times genes. To determine whether these different S. aureus isolates for nasal carriage of S. aureus over a 5 month period. The four induce different humoral antibody responses, anti-staphylococcal groups of twelve macaques were housed in separate rooms. For 9 antibody levels in sera of the 47 rhesus macaques were compared macaques (18.8%), all nasal culture results were negative. Eighteen to those in sera of 20 healthy human volunteers (Figure 5). macaques (37.5%) had one positive culture, 13 (27.1%) had two Significant differences in anti-staphylococcal IgG and IgA levels positive cultures, and for 8 macaques (16.7%) S. aureus was found between rhesus macaques and healthy volunteers were observed in all three cultures. for 29 and 25 out of 40 antigens, respectively (Table 4). In general, Isolates from these 48 duo-housed macaques were also typed by the median antibody serum levels in humans were higher than PFGE, and spa types were determined for representative isolates those in macaques. However, the median serum IgG level directed from each PFGE cluster (Figure 3). In macaques with more than against SSL1 and SSL5 was significantly higher in macaques than one S. aureus positive nasal culture, a strain replacement occurred in humans. The median serum IgA level directed against SSL9 in half of the animals. was significantly higher in macaques than in humans. It seemed that there was one most prevalent ‘‘epidemic’’ strain in each animal room. This strain spread from one or a couple of Association of anti-staphylococcal antibody levels with macaques to most others. This phenomenon was clear for rooms 1 presence of genes (PFGE type B, spa type t5013), 2 (PFGE type D, spa type t5016) To study whether the low antibody levels in rhesus macaques and 3 (PFGE type C9, spa type t5018), while for room 4, the were due to absence of the genes encoding the antigens, 10 isolates epidemic strain was less clear (PFGE type A, spa type t5013, or from the macaques that were screened three times, were selected PLoS ONE | www.plosone.org 8 October 2011 | Volume 6 | Issue 10 | e26170  Rhesus Macaques as Natural Hosts of S. aureus Figure 4. Relation between S. aureus nasal colonization and level of anti-staphylococcal IgG (A) and IgA (B). Antibody levels are reflected by Median fluorescence intensity (MFI) value. Each symbol represents a single rhesus macaque. Blue diamonds represent macaques without S. aureus positive culture, red triangles represent macaques with one positive culture, green squares represent macaques with two positive cultures, and black circles represent macaques with three positive cultures. Median values are indicated by horizontal lines. Arrows indicate statistically significant differences in median values (Mann-Whitney U test). More S. aureus positive cultures were not related to high – or low – anti- staphylococcal antibody levels. CHIPS, chemotaxis inhibitory protein of S. aureus; Clf, clumping factor; Efb, extracellular fibrinogen-binding protein; PLoS ONE | www.plosone.org 9 October 2011 | Volume 6 | Issue 10 | e26170  Rhesus Macaques as Natural Hosts of S. aureus ET, exfoliative toxin; Fnbp, fibronectin-binding protein; HlgB, c hemolysin B; Isd, iron-responsive surface determinant; Luk, leukocidin; SasG, S. aureus surface protein G; SCIN, staphylococcal complement inhibitor; Sdr, serine-aspartate dipeptide repeat protein; SE, staphylococcal enterotoxin; SSL, staphylococcal superantigen-like protein; TSST-1, toxic shock syndrome toxin 1. doi:10.1371/journal.pone.0026170.g004 for determination of the presence of genes encoding antigens human S. aureus isolates are underlined by major differences in against which a low MFI value was found in macaques, while MFI both anti-staphylococcal antibody levels and gene content of the S. values in humans were high (SCIN, CHIPS, ClfA, ClfB, Efb, aureus isolates between these two species. This indicates that new TSST-1, FnbpA, and FnbpB). Results are shown in Table 5. None host-specific lineages of S. aureus have now been found in rhesus of the isolates from rhesus macaques were scn-, tst-, or fnbB- macaques. positive, which explains why antibody levels were at background In contrast to the macaque S. aureus isolates, most human strains level. On the other hand, all 10 isolates were clfB- and efb-positive, harbour at least one gene encoding superantigens, on average 5 or and the macaques that carry these isolates had high antibody levels 6 genes, among which those encoding the egc superantigens SEG, against these antigens. Nine out of 10 S. aureus isolates did not SEI, SEM, SEN, and SEO are most prevalent [23,43,44]. These possess the gene encoding for CHIPS, which explains why the genes were also most prevalent in the macaque isolates. The MFI values were at background level. The macaque from which relatively low number of isolates containing superantigen genes the chp-positive strain was isolated also had MFI values at could also be due to the fact that the primers used in this study background level. Eight isolates were clfA-positive, with corre- were designed for S. aureus strains isolated from humans. sponding high MFI values for 6 macaques. Interestingly, the two Therefore, it can not be concluded that most macaque S. aureus macaques from which the clfA-negative strains were isolated, still isolates do not carry superantigen genes at all, but that, at least, if had high MFI values. Nine isolates possessed the gene encoding they are present, these genes differ from those found in human S. FnbpA, which corresponds with MFI values just above back- aureus isolates. ground level (range 22–616). However, the macaque with the fnbA- In addition, half of the macaque isolates could not be typed with negative S. aureus strain had MFI values above background level as the current agr multiplex PCR system. Of those that could be well. These differences are probably due to a previous exposure to typed, most belonged to agr types I or III, which is in contrast to an other S. aureus strain. human S. aureus isolates. These agr types are also the largest groups (agr type I 35% and type III 38%), but 25% of the isolates contains Discussion agr type II [45]. In contrast, agr type IV is hardly found among human isolates (2%), while 19% of the typeable macaque isolates Nasal carriage of S. aureus plays a key role in the epidemiology harboured this type. The agr untypeable S. aureus isolates probably and pathogenesis of staphylococcal infections [27,28]. Eradication have a deletion of the agr locus or extensive sequence variation at of S. aureus from the nose has proven to be effective in the the primer sites and could therefore not be typed. This further reduction of staphylococcal infections [29–32]. This indicates that emphasizes that S. aureus isolates in rhesus macaques differ from the anterior nasal region is a primary ecological reservoir of S. those in humans. aureus [33,34], although throat and perineum are important Next, we studied the persistence of nasal S. aureus carriage in 48 reservoirs as well [35–37]. However, nasal re-colonization may rhesus macaques. PFGE and spa typing of these isolates, as well as occur within weeks to months in those who have successfully been the absence of certain antigen genes while serum anti-staphylo- decolonized [38,39]. In order to develop new strategies in coccal antibody levels against these antigens were high, suggest prevention of staphylococcal disease, acquiring additional knowl- that persistent nasal S. aureus carriage as observed in humans is not edge about the underlying mechanisms of S. aureus nasal carriage is present among these rhesus macaques. Human persistent carriers important. Artificial inoculation of human volunteers offers are usually colonized by the same S. aureus strain over a long time opportunities to study these mechanisms [40]. While such semi- period [46], while half of the studied rhesus macaques carried clinical studies in humans remain the most informative, animal different S. aureus isolates, even over a 5 month period. Frequent models of S. aureus colonization sometimes enable a more detailed transmission of S. aureus isolates rather than persistent nasal investigation of the processes involved in pathogenesis by allowing carriage was suggested by the presence of one or two epidemic for more risky interventions. strains in each animal room. In the present study, we describe for the first time natural nasal The absence of persistent nasal carriage was underlined by the S. aureus carriage in rhesus macaques. The nasal cavity of these absence of an association between nasal carrier status and anti- macaques appears to be an important reservoir of S. aureus, as is staphylococcal antibody levels in serum of rhesus macaques, as was also observed in humans. In a single screening of their noses, 39% observed in humans. Differences in IgG levels between human of the 731 rhesus macaques had a positive culture, which is persistent carriers and non-carriers were reported for alpha comparable to the human situation with ,20% persistent carriers hemolysin, major autolysin, IsdA and IsdH, immunodominant and ,30% intermittent carriers [28,41,42]. Most of these S. aureus secretory antigen A (IsaA), major histocompatibility complex class isolates were different from human S. aureus isolates: rhesus II analogue protein w (Map-w), ClfB, TSST-1, and SEA. IgA macaque isolates formed separate PFGE clusters, 59% had one of levels were different between persistent carriers and non-carriers the 17 spa types that were not yet described, the previously for TSST-1, SEA, ClfA, and ClfB [14,47,48]. described spa types t189, t516, t729, t786, and t1814 were rare as In addition, the differences in anti-staphylococcal antibody well (Ridom SpaServer), in most macaque isolates genes encoding levels in rhesus macaques and humans, independent of nasal superantigens were not present, and half of the isolates could not carrier status, again underlined the differences between S. aureus be agr typed. Moreover, in the majority of macaque isolates new isolates in humans and those in macaques. The major immuno- STs were found which were not related to described STs. Three genic proteins in human S. aureus strains do not seem to elicit a isolates were part of CC45, and one was part of CC1. These humoral immune response in rhesus macaques. macaque isolates were therefore more comparable to human In conclusion, the present study demonstrates that rhesus isolates than the other ones. Differences between macaque and macaques are natural hosts of S. aureus, and that S. aureus isolates PLoS ONE | www.plosone.org 10 October 2011 | Volume 6 | Issue 10 | e26170  Rhesus Macaques as Natural Hosts of S. aureus Figure 5. Level of anti-staphylococcal IgG (A) and IgA (B) in rhesus macaques and human volunteers. Antibody levels are reflected by Median fluorescence intensity (MFI) value. Each symbol represents a single rhesus macaque or human. Blue squares represent healthy rhesus macaques and red diamonds represent healthy human volunteers. Median values are indicated by horizontal lines. Arrows indicate statistically significant differences in median values (Mann-Whitney U test). Anti-staphylcoccal IgG and IgA levels were significantly different between rhesus macaques and humans for 29 and 25 out of 40 antigens, respectively. CHIPS, chemotaxis inhibitory protein of S. aureus; Clf, clumping factor; Efb, extracellular fibrinogen-binding protein; ET, exfoliative toxin; Fnbp, fibronectin-binding protein; HlgB, c hemolysin B; Isd, iron-responsive surface determinant; Luk, leukocidin; SasG, S. aureus surface protein G; SCIN, staphylococcal complement inhibitor; Sdr, serine-aspartate dipeptide repeat protein; SE, staphylococcal enterotoxin; SSL, staphylococcal superantigen-like protein; TSST-1, toxic shock syndrome toxin 1. doi:10.1371/journal.pone.0026170.g005 PLoS ONE | www.plosone.org 11 October 2011 | Volume 6 | Issue 10 | e26170  Rhesus Macaques as Natural Hosts of S. aureus Table 4. Median fluorescence intensity (MFI) values reflecting antigen-specific IgG and IgA levels in rhesus macaques (RM) and healthy human volunteers (HV). MFI value, median (range) MFI value, median (range) S. aureus protein S. aureus protein (antibody (antibody isotype) RM HV Pa isotype) RM HV Pa alpha toxin (IgG) 14337 (7438–15574) 14587 (6355–17465) 0.285 SEA (IgG) 88 (17–3565) 3839 (29–11365) ,0.001 alpha toxin (IgA) 1540 (139–4729 1775 (215–9549) 0.154 SEA (IgA) 16 (1–449) 247 (5–9345) ,0.001 CHIPS (IgG) 20 (0–2580) 13310 (2933–16281) ,0.001 SEB (IgG) 34 (0–2836) 4223 (190–13622) ,0.001 CHIPS (IgA) 1 (0–130) 7494 (957–11238) ,0.001 SEB (IgA) 2 (0–673) 90 (2–398) ,0.001 ClfA (IgG) 454 (16–8250) 4029 (670–13449) ,0.001 SEC (IgG) 4551 (1–14006) 12720 (4713–15884) ,0.001 ClfA (IgA) 580 (4–6237) 673 (145–4730) 0.373 SEC (IgA) 280 (0–4896) 600 (63–3943) 0.004 ClfB (IgG) 1066 (65–7518) 7272 (2499–14127) ,0.001 SED (IgG) 84 (3–533) 800 (33–7924) ,0.001 ClfB (IgA) 1232 (0–6962) 2357 (255–10038) 0.125 SED (IgA) 1 (0–85) 19 (0–736) ,0.001 Efb (IgG) 622 (162–6325) 4238 (770–12855) ,0.001 SEE (IgG) 3 (0–229) 258 (7–3017) ,0.001 Efb (IgA) 69 (0–1575) 4775 (257–8125) ,0.001 SEE (IgA) 0 (0–151) 12 (0–1856) ,0.001 ETA (IgG) 156 (10–5670) 234 (16–14891) 0.217 SEG (IgG) 37 (0–3861) 517 (5–4385) 0.002 ETA (IgA) 30 (1–366) 19 (6–1127) 0.742 SEG (IgA) 5 (0–353) 22 (0–432) 0.035 ETB (IgG) 72 (0–10405) 55 (1–2855) 0.515 SEH (IgG) 1282 (0–10804) 1400 (9–15602) 0.468 ETB (IgA) 3 (0–1023) 20 (1–1529) 0.026 SEH (IgA) 88 (0–2083) 44 (0–2158) 0.821 FnbpA (IgG) 208 (20–3374) 1269 (222–6132) ,0.001 SEI (IgG) 4 (0–5154) 257 (0–3544) ,0.001 FnbpA (IgA) 73 (1–1047) 744 (26–5261) ,0.001 SEI (IgA) 0 (0–111) 8 (0–840) ,0.001 FnbpB (IgG) 111 (9–1089) 478 (54–2758) ,0.001 SEJ (IgG) 105 (5–7274) 2538 (95–16246) ,0.001 FnbpB (IgA) 15 (2–253) 253 (19–2603) ,0.001 SEJ (IgA) 1 (0–217) 25 (0–2236) ,0.001 HlgB (IgG) 8519 (2595–10365) 10761 (2979–13212) 0.001 SEM (IgG) 12 (0–7325) 307 (8–2738) ,0.001 HlgB (IgA) 474 (10–2799) 556 (44–6394) 0.233 SEM (IgA) 1 (0–354) 56 (0–1128) ,0.001 IsdA (IgG) 2811 (508–8602) 2665 (576–5183) 0.732 SEN (IgG) 7 (0–1166) 631 (0–2672) ,0.001 IsdA (IgA) 248 (13–3997) 357 (9–8058) 0.940 SEN (IgA) 0 (0–155) 7 (0–2743) ,0.001 IsdH (IgG) 929 (44–8170) 3921 (605–9420) ,0.001 SEO (IgG) 40 (7–2009) 306 (147–653) ,0.001 IsdH (IgA) 311 (3–4328) 294 (43–2117) 0.661 SEO (IgA) 3 (0–117) 20 (4–544) ,0.001 LukD (IgG) 1082 (1852–15031) 12226 (3781–16731) 0.011 SEQ (IgG) 5 (0–7125) 341 (0–3763) ,0.001 LukD (IgA) 641 (0–3418) 694 (106–4088) 0.753 SEQ (IgA) 0 (0–132) 19 (0–2219) ,0.001 LukE (IgG) 12386 (5270–14191) 12541 (5955–15590) 0.732 SER (IgG) 3 (0–1797) 63 (0–2654) ,0.001 LukE (IgA) 1181 (28–5431) 664 (38–7766) 0.524 SER (IgA) 0 (0–40) 9 (0–357) 0.001 LukF (IgG) 7321 (1240–12631) 10579 (2984–14996) 0.007 SSL1 (IgG) 8783 (1520–14022) 4683 (518–11914) 0.005 LukF (IgA) 321 (0–2554) 433 (38–3916) 0.041 SSL1 (IgA) 198 (0–4066) 195 (6–2947) 0.956 LukS (IgG) 10470 (1939–14385) 8734 (1662–16976) 0.135 SSL11 (IgG) 867 (43–8824) 1107 (122–8955) 0.311 LukS (IgA) 602 (0–6321) 1015 (13–10014) 0.524 SSL11 (IgA) 46 (3–1495) 96 (8–2162) 0.215 SasG (IgG) 89 (3–3376) 197 (3–2791) 0.093 SSL3 (IgG) 7280 (898–12949) 7813 (1618–15432) 0.403 SasG (IgA) 8 (0–5408) 33 (4–3950) 0.009 SSL3 (IgA) 321 (5–3062) 519 (24–5234) 0.244 SCIN (IgG) 187 (5–7259) 11333 (7572–15438) ,0.001 SSL5 (IgG) 4138 (621–9547) 3267 (511–5928) 0.021 SCIN (IgA) 14 (0–1220) 2945 (211–10036) ,0.001 SSL5 (IgA) 97 (6–1293) 115 (27–933) 0.264 SdrD (IgG) 233 (17–2844) 1683 (400–6924) ,0.001 SSL9 (IgG) 11968 (4788–13605) 9255 (1005–15161) 0.150 SdrD (IgA) 64 (3–2205) 634 (48–4758) ,0.001 SSL9 (IgA) 444 (35–3523) 138 (20–1158) 0.001 SdrE (IgG) 250 (28–6655) 2045 (251–8626) ,0.001 TSST-1 (IgG) 32 (4–9350) 12051 (0–16927) ,0.001 SdrE (IgA) 218 (0–3017) 604 (28–3650) 0.015 TSST-1 (IgA) 4 (0–125) 932 (10–3504) ,0.001 a Differences in antigen-specific MFI values between groups were considered to be statistically significant at P,0.05 (Mann-Whitney U test). doi:10.1371/journal.pone.0026170.t004 from rhesus macaques versus those from humans differ in many nasal cavity is an important reservoir in both species, which aspects. Therefore, rhesus macaques are not suitable for studying implies that the rhesus macaque provides an interesting model for S. aureus persistent carriage as is seen in humans. However, the studying short term nasal colonization, and in particular bacterial PLoS ONE | www.plosone.org 12 October 2011 | Volume 6 | Issue 10 | e26170  Rhesus Macaques as Natural Hosts of S. aureus Table 5. Association of anti-staphylococcal antibody levels with presence of genes in 10 S. aureus strains isolated from rhesus macaques. SCIN CHIPS ClfA ClfB MFI value, MFI value, MFI value, MFI value, MFI value, MFI value, MFI value, MFI value, Isolate no. Gene IgG IgA Gene IgG IgA Gene IgG IgA Gene IgG IgA 1 2 356 2 2 6 0 2 664 82 + 291 45 2 2 6404 77 2 23 1 + 2893 8252 + 6871 10067 3 2 5 3 2 15 1 + 3033 10785 + 997 4604 4 2 305 2 2 8 0 + 142 660 + 816 238 5 2 759 44 2 65 0 + 275 5213 + 1382 805 6 2 3702 26 + 173 3 2 282 4223 + 2423 9292 7 2 3067 127 2 30 2 + 26 7 + 866 462 8 2 492 11 2 886 0 + 27 37 + 2232 310 9 2 155 9 2 338 11 + 664 3549 + 462 549 10 2 27 76 2 8 27 + 17 1574 + 192 1574 Efb TSST-1 FnbpA FnbpB MFI value, MFI value, MFI value, MFI value, MFI value, MFI value, MFI value, MFI value, Isolate no. Gene IgG IgA Gene IgG IgA Gene IgG IgA Gene IgG IgA 1 + 1011 107 2 14 2 + 60 36 2 12 23 2 + 2332 2773 2 58 27 + 545 254 2 477 169 3 + 2564 402 2 180 9 + 181 100 2 1132 255 4 + 1156 101 2 10 0 + 157 56 2 78 86 5 + 373 48 2 43 16 + 165 616 2 63 223 6 + 279 321 2 37 0 + 192 544 2 161 300 7 + 416 25 2 30 22 + 131 22 2 324 13 8 + 679 61 2 37 1 + 508 134 2 149 74 9 + 3777 1069 2 197 5 + 74 51 2 24 35 10 + 2003 1061 2 8 4 2 356 502 2 26 263 doi:10.1371/journal.pone.0026170.t005 factors involved in adherence. Furthermore, rhesus macaque Dorothee Grumann, Suzan Rooijakkers, Jan-Ingmar Flock, and John provide an autologous system in which transmission of S. aureus Fraser for kindly supplying the S. aureus proteins. strains between individuals can be studied, and are therefore a useful model for studying infection prevention. Whether data from Author Contributions these studies can be directly extrapolated to humans is unclear. Conceived and designed the experiments: SB WJBW BO FAWV PJH However, possibilities for extrapolation from rhesus macaque to HAV AB. Performed the experiments: SB SVS BO CPV HAB XWH human appear reasonable because of their close evolutionary FAWV. Analyzed the data: SB WJBW SVS BO CPV HAB RJLW XWH. relatedness. Contributed reagents/materials/analysis tools: SB WJBW BO XWH IK PJH HAV AB. Wrote the paper: SB WJBW RJLW IK PJH HAV AB. Acknowledgments The authors wish to thank Deborah Horst-Kreft for her technical assistance. We thank Timothy Foster, Gerard Lina, Silva Holtfreter, References 1. Needham AJ, Kibart M, Crossley H, Ingham PW, Foster SJ (2004) Drosophila 5. van Belkum A, Melles DC, Peeters JK, van Leeuwen WB, van Duijkeren E, et al. melanogaster as a model host for Staphylococcus aureus infection. Microbiology 150: (2008) Methicillin-resistant and -susceptible Staphylococcus aureus sequence type 2347–2355. 398 in pigs and humans. Emerg Infect Dis 14: 479–483. 2. 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