Environmental Microbiology Reports (2012) 4(1), 141–146 doi:10.1111/j.1758-2229.2011.00316.x Highly divergent Staphylococcus aureus isolates from African non-human primates emi4_316 F. Schaumburg,1,3* A. S. Alabi,3,4 R. Köck,2 A. Mellmann,2 P. G. Kremsner,3,4 C. Boesch,5 K. Becker,1 F. H. Leendertz6 and G. Peters1 Institutes of 1Medical Microbiology and 2Hygiene, University Hospital Münster, Münster, Germany. 3 Medical Research Unit, Albert Schweitzer Hospital, Lambaréné, Gabon. 4 Institute of Tropical Medicine, University Hospital Tübingen, Tübingen, Germany. 5 Department of Primatology, Max-Planck-Institute for Evolutionary Anthropology, Leipzig, Germany. 6 Research Group Emerging Zoonoses, Robert-Koch-Institut, Berlin, Germany. Summary Staphylococcus aureus is a bacterium that colonizes and infects both humans and animals. As little is known about the phenotypic and molecular characteristics of S. aureus from wild animals in subSaharan Africa, the objective of the study was to characterize S. aureus isolates from wildlife and to analyse if they differed from those found among humans. The resistance to penicillin was low in S. aureus isolates from non-human primates (2.9%). Phylogenetic analysis based on the concatenated sequences from multilocus sequence typing revealed two highly divergent groups of isolates. One group was predominated by S. aureus that belonged to known human-related STs (ST1, ST9 and ST601) and mainly derived from great apes. A second clade comprised isolates with novel STs. These isolates were different from classical human S. aureus strains and mainly derived from monkeys. Our findings provide the basis for future studies addressing the inter- and intra-species transmission of S. aureus in Africa. Introduction Staphylococcus aureus is one of the most frequent causes of superficial skin and deep-seated infections in humans. It Received 25 August, 2011; accepted 14 November, 2011. *For correspondence. E-mail frieder.schaumburg@ukmuenster.de; Tel. (+49) 251 83 52752; Fax (+49) 251 83 52768. Funding: The study was funded by the Deutsche Forschungsgemeinschaft (DFG, EI 247/ 8-1, FR 2569/2-1, LE 1813/4-1). © 2011 Society for Applied Microbiology and Blackwell Publishing Ltd 141..146 has been demonstrated that many mammals, reptiles and birds can also be colonized and infected by S. aureus (Cuny et al., 2010). Most recently, the impact of S. aureus as a zoonotic pathogen has been highlighted. Exemplarily, in many western countries industrially raised livestock (pigs, veal calves, poultry) was found to represent a significant reservoir of methicillin-resistant S. aureus and a major source of human methicillin-resistant S. aureus colonization and infections in regions characterized by a high density of livestock farming (Köck et al., 2010). The transmission of S. aureus between humans and animals can be bidirectional as S. aureus isolates can also be transmitted from human to animals (i.e. poultry) (Lowder et al., 2009). Whereas ‘host switching’ of S. aureus has been intensively studied in industrialized countries, nothing is known about interspecies transmission and its public health impact in developing regions. Close contact of humans to wild animals occurs during butchering, trade and consumption of bushmeat and may facilitate a transmission of potential pathogens between humans and animals (Wolfe, 2005; Leendertz et al., 2006; Jones et al., 2008). Bushmeat is meat from wild animals living in the African rainforest or savannah (Barnes, 2002). To provide the basis for future investigations into inter- and intra-species transmission of S. aureus in Africa, we characterized the susceptibility to antibiotic agents, genotypes and virulence patterns of S. aureus isolates from African non-human primates, which are important targets for hunters and whose meat is frequently consumed in sub-Saharan regions. Results and discussion Animal population Samples were derived from monkeys (n = 64) and great apes (n = 31, Table S1). One Cercopithecus nictitans from Gabon and two Piliocolobus badius from Côte d’Ivoire carried two genotypically different S. aureus isolates. Five samples from fruit wadges from chimpanzees in Côte d’Ivoire harboured two different S. aureus isolates and one fruit wadge carried three different isolates. Fruit wadges are the spit out remains of forest fruits, chewed by chimpanzees for several minutes to suck out the juice. All S. aureus isolates (n = 58) were coagulase-positive, showed a typical biochemical profile and were confirmed as S. aureus by 16S rRNA gene sequencing. 142 F. Schaumburg et al. Antimicrobial susceptibility In total, 57 isolates (98.3%) were susceptible to penicillin. The beta-lactamase encoding gene blaZ was only detected in the penicillin-resistant isolate. This isolate derived from a chimpanzee from Côte d’Ivoire. All isolates were susceptible to methicillin (as confirmed by the absence of mecA), aminoglycosides, fluoroquinolones, macrolides, lincosamides (including inducible clindamycin resistance), nitrofurantoin, fosfomycin, rifampicin, tetracycline, cotrimoxazole and vancomycin. Penicillin resistance is rare in S. aureus isolates from remote African regions, even in humans who are more likely to be exposed to antibiotics (Schaumburg et al., 2011a). The penicillin resistance could be due to a treatment of chimpanzees with respiratory tract infections in Taï National Parc, Côte d’Ivoire with benzathine benzylpenicillin, a slowly absorbed penicillin. Molecular typing Sequence based typing of the hypervariable region of S. aureus protein A (spa-typing) resulted in 32 different spa types (Table S2). Two spa types were found in two different species: t127 (Cercopithecus polykomos, Pan troglodytes, both from Côte d’Ivoire) and t6533 (Cercopithecus cephus and C. nictitans, both from Gabon) indicating a transmission between these species. Chimpanzees hunt and consume other monkeys such as Colobus sp. and Cercopithecus sp. (Boesch and Boesch, 1989). This might explain a transmission of S. aureus from prey to hunters as it has been already shown for various viruses (Leendertz et al., 2004; 2008). To analyse the phylogenetic relatedness of the S. aureus isolates, we used the concatenated sequences of the seven multilocus sequence typing (MLST) housekeeping genes of each ST and constructed a NeighborJoining tree (Fig. 1). All sequences clustered into two groups and the mean distance between the two groups was 0.085 base substitutions per site. The split into two groups was supported by a bootstrap value of 100. The majority of S. aureus isolates from group 1 derived from great apes (n = 24, 72.7%) and clustered with ST8, ST15, ST36 and ST152, which are well known ST from human S. aureus isolates. This might mirror the co-divergence of S. aureus and its primate host and might reflect the close phylogenetic relation between chimpanzees and humans. In contrast, isolates from group 2 mainly originated from monkeys (n = 14, 93.3%) and clustered with the ST of a divergent isolate recently found in a human S. aureus carrier from rural Gabon (ST1822) (Schaumburg et al., 2011b) and the known divergent ST1223 (Ruimy et al., 2009). Central African Pygmies, who frequently hunt and consume bushmeat, are not colonized with isolates belonging to the divergent clade, which argues against a frequent transmission of theses isolates from animals to humans. Therefore, targeted studies are needed to investigate the transmissibility of animal-adapted strains to humans. Despite its divergent position, STs of group 2 are phylogenetically closer to group 1 than to Staphylococcus simiae the closest related coagulase-negative species of S. aureus (Fig. 1). Virulence factors and capsular types In total, 18 virulence factors were tested (Table S3). The median number (range) of virulence genes was 2 (1–5) per isolate, which was markedly lower than in isolates from remote Gabonese Pygmies (2 vs. 3.3) who share the same habitat with many animals species of this study (Schaumburg et al., 2011a). Pyrogenic toxin superantigens were encoded by 26 (44.8%) isolates with a predominance of sei (29.3%), seh and seg (19.0% each) (Table S2). The PantonValentine leukocidin (PVL) encoding genes were found in isolates from C. nictitans (ST1855, Gabon) and P. troglodytes (ST 1928, Côte d’Ivoire, Table S3). Other virulence genes were not detected (sea, sed, see, sej, tst, eta, edin-A, edin-C). PVL is an important pore forming protein toxin that is frequently associated with abscesses. S. aureus harbouring PVL seem to be endemic in humans in Africa (Gillet et al., 2002; Breurec et al., 2011; Schaumburg et al., 2011a,b). In contrast, the low prevalence of PVL in non-human primates suggests a different selection pressure for PVL-positive isolates. Because monkey cells have a reduced susceptibility to PVL our finding argues against a selective advantage of PVL-positive isolates in terms of clonal spread, if the host is not susceptible to this toxin (Olsen et al., 2010). Capsular polysaccharides (CP) are virulence determinants as they impede phagocytosis by monocytes and macrophages. The genes encoding CP5 (n = 21, 36.2%) and CP8 (n = 20, 34.5%) were equally distributed, 17 (24.6%) isolates were CP non-typeable. Concluding remarks African monkeys can be colonized by highly divergent S. aureus isolates, which are rarely found in humans while great apes mainly carry human-related S. aureus lineages. The characterization of these isolates is the basis for future investigations into inter- and intra-species transmission of S. aureus in sub-Saharan Africa. © 2011 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology Reports, 4, 141–146 Staphylococcus aureus from African animals 143 Fig. 1. Phylogenetic relation of S. aureus from African wildlife, S. simiae and S. haemolyticus. A Neighbor-Joining tree was constructed using the concatenated sequences of the seven S. aureus MLST housekeeping genes. Colours indicate the country of origin of the isolate; the name of the species from which the isolate derived is shown in italics; total numbers of isolates per ST are shown in brackets. Experimental procedures Sample collection From Gabon, samples originated from 45 monkeys and one gorilla sold for consumption at a commercial market or on the roadside in the province ‘Moyen Ogooué’ (Table S1). Nasal swabs were taken within 6–12 h after death of the animals. In Côte d’Ivoire, nasal swabs were taken from 10 red colobus monkeys (P. badius) and 10 black and white colobus monkeys (C. polykomos) anaesthetized in the course of a primate health study in the Taï National Park, Côte d’Ivoire © 2011 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology Reports, 4, 141–146 144 F. Schaumburg et al. (Leendertz et al., 2008; 2010). Furthermore, fruit wadges were collected from 30 chimpanzees habituated to human presence in the same area of the Taï National Park (Boesch and Boesch, 2000). All animals in this study were considered to be wild and have never been touched by humans. Identification and antimicrobial susceptibility testing All samples were streaked on SAID and blood agar plates (bioMérieux, Marcy l’Etoile, France). Presumptive S. aureus colonies were identified by colony characteristics, catalase, coagulase (Staph-ase, bioMérieux), and latex agglutination test (Pastorex Staph-Plus, Bio-Rad Laboratories, Marnes-laCoquette, France). Biochemical species confirmation and antimicrobial susceptibility testing were performed using Vitek2 automated systems (bioMérieux). All isolates were subjected to ribosomal 16S rRNA gene sequencing for genotypic species confirmation (Becker et al., 2004b). To confirm susceptibility to penicillin and methicillin, we performed a PCR targeting the blaZ and mecA genes respectively (Becker et al., 2006; Kaase et al., 2008). et al., 2009). As a reference for common STs found among carrier isolates from sub-Saharan Africa we selected ST8, ST15; ST36 and ST152 (Ruimy et al., 2008; Schaumburg et al., 2011b). To refer to known divergent isolates we chose ST1223 (Ruimy et al., 2009) and ST1822 (Schaumburg et al., 2011b). The evolutionary divergence of the concatenated sequences was calculated using the Maximum Composite Likelihood method. Ethics The non-invasive samples from animals were collected in accordance with international guidelines and under the permission of the national authorities (OIPR, Office Ivoirienne des Parcs et Reserves). Ethical approval for samples from bushmeat was not required as samples were taken from dead animals only. The investigators did not have any influence on the amount and the animal species that were hunted. Statistics Virulence factors We used multiplex PCR approaches to detect genes encoding the pyrogenic toxin superantigens including toxic shock syndrome toxin (tst), enterotoxins (sea, seb, sec, sed, see, seg, seh, sei, sej), the PVL (lukS-PV/lukF-PV), the exfoliative toxins (eta, etb, etd), and members of the epidermal cell differentiation inhibitor (edin-A, edin-B, edin-C) (Becker et al., 2004a; von Eiff et al., 2004). Molecular typing All isolates were spa typed based on the repeat pattern of the hypervariable region of the S. aureus protein A (spa) gene (Mellmann et al., 2006). Multilocus sequence typing was performed exemplarily for one isolate of each spa type (Enright et al., 2000). Alternative primers were used for aroE and glpF (Ng et al., 2009; Ruimy et al., 2009). Subtypes of the accessory gene regulator (agrI–IV) and CP types 5 and 8 were analysed by multiplex PCR (von Eiff et al., 2004; Goerke et al., 2005). Odds ratio and the 95% confidence interval were calculated to analyse the association between categorical variables. Significance of association was analysed using chi-squared test or Fisher’s exact test. Statistical analysis was performed using the software ‘R’ (http://cran.r-project.org, Version: 2.10.1) and package ‘epicalc’. Acknowledgement We thank the Ivorian authorities (Ministry of Environment and Forests, Ministry of Research, the directorship of the Taï National Park, the Office Ivoirien des Parcs et Réserves and the Swiss Research Centre in Abidjan) for their long-term support. We also thank S. Schenk, S. Metzger, S. Köndgen and B. Biallas for the sample collection in Côte d’Ivoire and Gabon. We thank S. A. J. Leendertz and W. Rietschel for assisting colobus captures. We are grateful to B. Grünastel for microbiological cultures, U. Keckevoet and I. Ramminger for spa typing and A. Hassing and M. Schulte for the detection of virulence factors. For helpful discussion and copy editing we thank the Research Group ‘Emerging Zoonoses’ from RKI. Phylogenetic analysis All MLST STs of this study were assigned to known clonal complexes by the eBURST algorithm using the whole MLST dataset and the stringent group definition of six out of seven shared identical alleles (http://saureus.mlst.net). Related spa types were identified and clustered into spa clonal complexes (spa-CC) using the BURP-algorithm (Staph Type 2.1.1 software, Ridom GmbH, Münster, Germany) with preset parameters as published (Mellmann et al., 2007). Phylogenetic analyses were performed with MEGA4 (http:// www.megasoftware.net) using the concatenated sequences of seven MLST genes and the Neighbor-Joining method. We did not include arcC in the concatenated sequences of S. simiae due to difficulty in amplifying this gene (Ruimy References Barnes, R.F.W. (2002) The bushmeat boom and bust in West and Central Africa. Oryx 36: 236–242. Becker, K., Friedrich, A.W., Peters, G., and von Eiff, C. (2004a) Systematic survey on the prevalence of genes coding for staphylococcal enterotoxins SEIM, SEIO, and SEIN. Mol Nutr Food Res 48: 488–495. Becker, K., Harmsen, D., Mellmann, A., Meier, C., Schumann, P., Peters, G., and von Eiff, C. (2004b) Development and evaluation of a quality-controlled ribosomal sequence database for 16S ribosomal DNA-based identification of Staphylococcus species. J Clin Microbiol 42: 4988–4995. © 2011 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology Reports, 4, 141–146 Staphylococcus aureus from African animals 145 Becker, K., Pagnier, I., Schuhen, B., Wenzelburger, F., Friedrich, A.W., Kipp, F., et al. (2006) Does nasal cocolonization by methicillin-resistant coagulase-negative staphylococci and methicillin-susceptible Staphylococcus aureus strains occur frequently enough to represent a risk of false-positive methicillin-resistant S. aureus determinations by molecular methods? J Clin Microbiol 44: 229– 231. Boesch, C., and Boesch, H. (1989) Hunting behavior of wild chimpanzees in the Taï National Park. Am J Phys Anthropol 78: 547–573. Boesch, C., and Boesch, H. (2000) The Chimpanzees of the Taï-Forest: Behavioural Ecology and Evolution. Oxford: Oxford University Press. Breurec, S., Fall, C., Pouillot, R., Boisier, P., Brisse, S., Diene-Sarr, F., et al. (2011) Epidemiology of methicillinsusceptible Staphylococcus aureus lineages in five major African towns: high prevalence of Panton-Valentine leukocidin genes. Clin Microbiol Infect 17: 633–639. Cuny, C., Friedrich, A., Kozytska, S., Layer, F., Nübel, U., Ohlsen, K., et al. (2010) Emergence of methicillin-resistant Staphylococcus aureus (MRSA) in different animal species. Int J Med Microbiol 300: 109–117. von Eiff, C., Friedrich, A.W., Peters, G., and Becker, K. (2004) Prevalence of genes encoding for members of the staphylococcal leukotoxin family among clinical isolates of Staphylococcus aureus. Diagn Microbiol Infect Dis 49: 157–162. Enright, M.C., Day, N.P., Davies, C.E., Peacock, S.J., and Spratt, B.G. (2000) Multilocus sequence typing for characterization of methicillin-resistant and methicillin-susceptible clones of Staphylococcus aureus. J Clin Microbiol 38: 1008–1015. Gillet, Y., Issartel, B., Vanhems, P., Fournet, J.C., Lina, G., Bes, M., et al. (2002) Association between Staphylococcus aureus strains carrying gene for Panton-Valentine leukocidin and highly lethal necrotising pneumonia in young immunocompetent patients. Lancet 359: 753–759. Goerke, C., Esser, S., Kummel, M., and Wolz, C. (2005) Staphylococcus aureus strain designation by agr and cap polymorphism typing and delineation of agr diversification by sequence analysis. Int J Med Microbiol 295: 67–75. Jones, K.E., Patel, N.G., Levy, M.A., Storeygard, A., Balk, D., Gittleman, J.L., and Daszak, P. (2008) Global trends in emerging infectious diseases. Nature 451: 990–993. Kaase, M., Lenga, S., Friedrich, S., Szabados, F., Sakinc, T., Kleine, B., and Gatermann, S.G. (2008) Comparison of phenotypic methods for penicillinase detection in Staphylococcus aureus. Clin Microbiol Infect 14: 614–616. Köck, R., Becker, K., Cookson, B., van Gemert-Pijnen, J.E., Harbarth, S., Kluytmans, J., et al. (2010) Methicillinresistant Staphylococcus aureus (MRSA): burden of disease and control challenges in Europe. Euro Surveill 15: 19688. Leendertz, F.H., Junglen, S., Boesch, C., Formenty, P., Couacy-Hymann, E., Courgnaud, V., et al. (2004) High variety of different simian T-cell leukemia virus type 1 strains in Chimpanzees (Pan troglodytes verus) of the Taï National Park, Cote d’Ivoire. J Virol 78: 4352–4356. Leendertz, F.H., Pauli, G., Maetz-Rensing, K., Boardman, W., Nunn, C., Ellerbrok, H., et al. (2006) Pathogens as drivers of population declines: the importance of systematic monitoring in great apes and other threatened mammals. Biol Conserv 131: 325–337. Leendertz, F.H., Zirkel, F., Couacy-Hymann, E., Ellerbrok, H., Morozov, V.A., Pauli, G., et al. (2008) Interspecies transmission of simian foamy virus in a Natural Predator-Prey System. J Virol 82: 7741–7744. Leendertz, S.A., Junglen, S., Hedemann, C., Goffe, A., Calvignac, S., Boesch, C., and Leendertz, F.H. (2010) High prevalence, coinfection rate, and genetic diversity of retroviruses in Wild Red Colobus Monkeys (Piliocolobus badius badius) in Taï National Park, Côte d’Ivoire. J Virol 84: 7427–7436. Lowder, B.V., Guinane, C.M., Ben Zakour, N.L., et al. (2009) Recent human-to-poultry host jump, adaptation, and pandemic spread of Staphylococcus aureus. Proc Natl Acad Sci USA 106: 19545–19550. Mellmann, A., Friedrich, A.W., Rosenkotter, N., Rothganger, J., Karch, H., Reintjes, R., and Harmsen, D. (2006) Automated DNA sequence-based early warning system for the detection of methicillin-resistant Staphylococcus aureus outbreaks. PLoS Med 3: e33. Mellmann, A., Weniger, T., Berssenbrugge, C., Rothganger, J., Sammeth, M., Stoye, J., and Harmsen, D. (2007) Based Upon Repeat Pattern (BURP): an algorithm to characterize the long-term evolution of Staphylococcus aureus populations based on spa polymorphisms. BMC Microbiol 7: 98. Ng, J.W., Holt, D.C., Lilliebridge, R.A., Stephens, A.J., Huygens, F., Tong, S.Y., et al. (2009) Phylogenetically distinct Staphylococcus aureus lineage prevalent among indigenous communities in northern Australia. J Clin Microbiol 47: 2295–2300. Olsen, R.J., Kobayashi, S.D., Ayeras, A.A., Ashraf, M., Graves, S.F., Ragasa, W., et al. (2010) Lack of a major role of Staphylococcus aureus Panton-Valentine Leukocidin in lower respiratory tract infection in nonhuman primates. Am J Pathol 176: 1346–1354. Ruimy, R., Maiga, A., Armand-Lefevre, L., Maiga, I., Diallo, A., Koumare, A.K., et al. (2008) The carriage population of Staphylococcus aureus from Mali is composed of a combination of pandemic clones and the divergent PantonValentine leukocidin-positive genotype ST152. J Bacteriol 190: 3962–3968. Ruimy, R., Armand-Lefevre, L., Barbier, F., Ruppe, E., Cocojaru, R., Mesli, Y., et al. (2009) Comparisons between geographically diverse samples of carried Staphylococcus aureus. J Bacteriol 191: 5577–5583. Schaumburg, F., Köck, R., Friedrich, A.W., Soulanoudjingar, S., Ateba Ngoa, U., von Eiff, C., et al. (2011a) Population structure of Staphylococcus aureus from remote African Babongo Pygmies. PLoS Negl Trop Dis 5: e1150. Schaumburg, F., Ngoa, U.A., Kösters, K., Köck, R., Adegnika, A.A., Kremsner, P.G., et al. (2011b) Virulence factors and genotypes of Staphylococcus aureus from infection and carriage in Gabon. Clin Microbiol Infect 17: 1507– 1513. Wolfe, N.D. (2005) Bushmeat hunting, deforestation, and prediction of zoonotic disease emergence. Emerg Infect Dis 11: 1822–1827. © 2011 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology Reports, 4, 141–146 146 F. Schaumburg et al. Supporting information Additional Supporting Information may be found in the online version of this article: Table S1. Characteristics of African wildlife. Table S2. Characteristics of S. aureus genotypes from bushmeat and wild-living African animals. Table S3. Distribution of virulence factors among S. aureus isolates from African animals. Please note: Wiley-Blackwell are not responsible for the content or functionality of any supporting materials supplied by the authors. Any queries (other than missing material) should be directed to the corresponding author for the article. © 2011 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology Reports, 4, 141–146