Microarray procedures

All confirmed S. aureus isolates were characterized using the StaphyType DNA microarray or S. aureus Genotyping Kits 2.0 kit (Alere Technologies GmbH, Jena, Germany). This array simultaneously detects 333 S. aureus target sequences, including species markers, antimicrobial resistance and virulence-associated genes, and SCCmec-associated genes and typing markers allowing isolates to be assigned to MLST sequence types (STs) and/or CCs, and SCCmec types. The latter kit also detects SCCmec XI/mecC [39] and it was applied to all isolates tested after 2013, to all CC130 and CC599 isolates as well as to select additional isolates (see S1 Table). Protocols and procedures as well as primer and probe sequences have been previously described in detail [39,109,110]. In brief, S. aureus isolates were stored frozen in commercially available cryotubes (various brands; at -40 or -80°C), and grown on Columbia blood agar and incubated overnight at 37°C. Bacterial cells were enzymatically lysed prior to DNA preparation using commercially available spin columns (Qiagen, Hilden, Germany). Purified DNA samples were used as templates in a linear primer elongation using one primer per target. All targets were amplified simultaneously, and within this step, biotin-16-dUTP was incorporated into the resulting single-stranded amplicons. Amplicons were stringently hybridized to the microarray followed by washing and an addition of a horseradish-peroxidase-streptavidin conjugate. After further incubation and washing, hybridizations were visualized by adding a locally precipitating dye. An image of the microarray was taken and analyzed using a designated reader, software and database.

PCRs for characterizing SCCmec XI

SCCmec XI was further characterized by PCR in three isolates (two CC130 isolates [39] and one ST599 isolate). PCR amplification targeted fragments across the entire element using overlapping primers [87] and the Expand long-template PCR System (Roche Diagnostics GmbH, Lewes, East Sussex, United Kingdom). Sizes of the resulting amplicons were compared to those of the mecC-positive reference strain M10/0061 [87].

MLST and spa

S. aureus MLST was performed for isolates which initially could not be identified based on their array hybridization profiles as well as for some isolates that have been discussed in separate studies [30; 39; 77; 107;108]. MLST was performed according to standard protocol [111] using the tools and database provided on the S. aureus MLST website (http://saureus.mlst.net/). Novel profiles were submitted to the MLST database.

Spa typing was performed according to previously published protocols [112] and sequences were analyzed using RIDOM nomenclature (http://www.spaserver.ridom.de/).

MLST and spa data are shown in S1 Table.

Clonal complex 1

Five CC1-MSSA isolates were identified, two from fallow deer, two from mouflon, and one from a red fox. They did not harbor mecA, mecC or any other resistance genes. Virulence associated markers included seh (but no other enterotoxin genes). None of the isolates had evidence for the presence of lysogenic beta-hemolysin converting phages as all isolates were hlb-positive and were negative for sea, see, sep, chp, sak and scn.

Two CC1-MRSA-IV isolates were identified from Austrian rooks [77] that carried blaZ, tetK, apha3 and sat (kana-/neomycin and streptothricin resistance).

CC1 is a common lineage among humans including both MSSA and community-acquired (CA-) MRSA; and CC1 is also frequently found in cattle [114,115]. A strain that closely resembled the MRSA from rooks was frequently found among humans in Romania [116].

Clonal complex 5

Three CC5-MSSA were identified, one from a Swedish grey partridge and two from hares from the island of Pellworm. The two hare isolates carried sea and the immune evasion complex (IEC) genes scn, chp and sak, whereas the partridge isolate lacked these genes. Genes encoding other enterotoxins or PVL were absent. None of the resistance genes covered was detected. CC5 is a common and widespread lineage in humans. It has recently [58] spread into domestic poultry (see Introduction) where it is now be commonly encountered [61]. A further dissemination to wild fowl such as partridges appears plausible.

Clonal complex 6

A single CC6-MSSA isolate was identified in a Swedish red fox. The hybridization pattern of the isolate was identical to the predicted hybridization pattern for clinical CC6-MSSA isolates 394_SAUR (GenBank JVIV) and C9 (GenBank LDVH). It harbored the beta-lactamase operon as well as the IEC genes sea, sak and scn.CC6 has been recovered occasionally from humans including butchers [117] and it has been implicated in episodes of food-poisoning [118]. It was previously identified in camels from Dubai, where it carried a different sea allele [21], as well as in non-human primates and various domestic animals [4,119].

Clonal complex 7

A single CC7-MSSA isolate was recovered from an Austrian red fox. It carried the IEC genes sea-N315, scn and sak genes. The isolate was indistinguishable from previously described human CC7-MSSA isolates [120,121].

Clonal complex 8

Several CC8-MSSA isolates were identified in Austrian animals including a red fox, a marmot and a mouflon. The fox isolate lacked antimicrobial resistance genes but harbored the sak and scn genes. The other two isolates were positive for blaZ, enterotoxins sed, sej, ser, as well as for the IEC genes sea, sak and scn. CC8 is a common lineage among humans [120,121] and several MRSA clones [109] originated from that lineage. It is not typically considered a zoonotic lineage although CC8-MRSA have been found in horses [24], wildlife [122] and whales [53].

Clonal complex 9

A single CC9-MSSA isolate was recovered from an Austrian wild boar. The isolate was positive for the egc locus but lacked other relevant toxin genes. It was positive for the tetracycline resistance gene tet(K) but other antimicrobial resistance genes were absent. CC9-MSSA has been found in wild chimpanzees in Ivory Coast [54] but we are not aware of any reports from European wildlife. However, CC9 livestock-associated MRSA are frequently associated with domestic pigs (see Introduction) so that a presence of CC9-MSSA in wild boar is perhaps not surprising.

Clonal complex 12

A single CC12-MSSA isolate was recovered from a Swedish harbor porpoise. The beta-lactamase operon (blaZ/R/I) was present, enterotoxin genes and IEC genes were absent, although they are frequently but not always found in human isolates of that lineage [120,121]. Apart from humans, CC12 has previously been found in chickens [60] and swine [123]. S. aureus has previously been reported in porpoises (see Introduction), but isolates belonged to other sequence types some of which were unique (CC1762), while others are known to be associated with humans or terrestrial mammals [48].

Clonal complex 15

Two isolates of CC15-MSSA were identified in an Austrian rook and a Swedish moose. They were essentially identical to previously characterized human isolates [120,121]. As in these human isolates, the sak gene was absent although, scn and chp were detected. Furthermore, the isolates lacked enterotoxin and PVL genes or lukM/lukF-P83. The beta-lactamase gene was present. CC15 is a widespread and common human lineage [120,121]. It has occasionally been reported from companion animals [124], primates [119] and mice [36].

Clonal complex 22

A single CC22-MSSA isolate was found in a fox from Germany. It harbored blaZ as well as the IEC genes chp, scn and sak. Essentially it was indistinguishable from previously described human isolates. Three CC22-MRSA isolates from Austrian rooks have previously been described [77]. In short, they carried PVL genes as well as blaZ, aacA-aphD, aadD and dfrS1. CC22 is a common “human lineage” but has also been found in companion animals with close contacts to humans [125,126] and in wild roe deer [11]. CC22 MRSA are extremely common in Western Europe, but the rook isolates were not identical to the ubiquitous Barnim/UK-EMRSA-15 epidemic strain differing in SCCmec subtype (IVc rather than IVh; [77]) and presence of PVL genes. They were identical to a strain observed in several Middle Eastern countries and people with Middle Eastern travel history [109,127,128,129,130].

Clonal complex 25

A single isolate of CC25-MSSA was identified from a German badger. It carried the blaZ operon, the enterotoxin B gene, edinB, etD and IEC genes sak, scn and chp. It was essentially identical to human isolates as previously characterized [120,121].

Clonal complex 30

One isolate of CC30-MSSA was found in an Austrian marmot. It carried the leukocidin genes lukM/lukF-P83. Surprisingly, it was positive for the multidrug resistance gene cfr that confers the PhLOPSa resistance phenotype, i.e., resistance to phenicols, lincosamides, oxazolidiones, pleuromutulin and streptogramin A compounds, and the phenicol resistance gene fexA. It was resistant to the oxazolidinone agent linezolid (MIC > 8 μg/mL) as well as to clindamycin and chloramphenicol. Another isolate originated from Austrian deer. It only harbored blaZ.

CC30 is a common human lineage [120,121,131,132] from which several MRSA [109] and toxigenic strains evolved [133,134]. CC30 has previously been found in animals, including camels [21] and pigs [82]. It also has been detected in Spanish white storks [66] and a Portuguese buzzard [68].

Clonal complex 49

CC49-MSSA isolates were recovered from two fecal samples of voles from Bavaria and from a road-killed wildcat from North Rhine-Westphalia. They carried leukocidin genes lukM/lukF-P83, but lacked enterotoxin genes. These animal isolates differed from previously described human isolates [120,132,135] and one genome sequence (Tager 104, GenBank AVBR [136]) in the presence of lukM/lukF-P83 and the absence of IEC genes associated with beta-hemolysin converting phages. They lacked any antimicrobial resistance or SCCmec associated markers.

This CC has been found in humans [120,132,135] but appears to be rare. Previously, CC49-MSSA have also been observed to cause an outbreak among red squirrels on the Isle of Wight and the island of Jersey that died from exudative, ulcerative dermatitis and superficial staphylococcal pyoderma [34]. This squirrel strain was found to be lukM-positive [33] like the isolates described in the present study, but unlike the human isolates mentioned above. This might indicate host specific adaptions related to the presence of a phage. CC49-MRSA-V has been observed in Swiss livestock [137] and CC49-MRSA-XI have been sequenced (NCBI BioSamples SAMEA1463364, SAMEA1463346).

Clonal complex 59

One CC59-MSSA was isolated from a shot Thuringian wild boar. The isolate carried erm(C) and blaZ but lacked enterotoxin and PVL genes or lukF-P83/lukM. It carried IEC genes chp and scn but sak was not detected. CC59 is widespread in humans and several MRSA strains originate from this lineage [109,138,139,140]. It has been detected in workers in contact with livestock or animal carcasses [141,142] and retail food [143] as well as in pets [144] but we are not aware of reports to date from wildlife.

Clonal complex 88

Three isolates of CC88-MSSA were identified from Austrian rooks. They lacked relevant toxin genes. All three harbored blaZ and the cat gene (encoding chloramphenicol resistance). CC88 is a widespread human lineage from which MRSA strains evolved that are common in Australia [145] and Sub-Saharan Africa [119,146,147,148,149]. It has been described from laboratory mice [35], from retail food [143] and from pigs [150].

Clonal complex 97

Twenty isolates of CC97-MSSA from Swedish wildlife were identified. One originated from a golden eagle, four from roe deer and the rest were from moose. Two isolates were found in roe deer from Germany, one suffering from botfly infestation, pyogranulomatous dermatitis and pneumonia, the other one from necrotic/gangrenous spondylodiscitis and pericarpitis. Two additional isolates originated from Austrian wild boar. All these isolates appeared identical to the sequenced reference strain Newbould 305 (GenBank AKYW, a bovine strain from Canada). They did not carry relevant resistance or toxin genes. The two Austrian isolates carried an isolated recombinase homologue ccrAA and reacted with new experimental probes (opp3B; ACSQ01000050.1 [4287:4313] and opp3C-C427 ACSQ01000050.1 [5150:5174]; [129]) indicating the presence of a type 3 ACME element.

CC97 has been reported from roe deer before, from the Italian Alps [11]. Besides, CC97 is frequently detected in humans [120], pigs [123,151], sheep [152] as well as in cattle [2,152,153], with the latter being phylogenetically related to deer species. Given the phylogenetic relationship between cervids and bovids, it can be postulated that this lineage is transmitted between both, or that it co-evolved with them. The eagle mentioned above might have been infected when scavenging.

Clonal complex 130

One CC130-MSSA was found in a fecal sample of a brown rat having the MLST profile ST2024 and the spa type t8403. Its array hybridization profile was essentially identical to predicted patterns for the sequenced strains O11 (GenBank AEUQ) and O46 (GenBank AEUR). It carried lukF-P83/lukM as well as edinB and etD2 [39]. It differed from O11 and O46 in the presence of cadmium resistance genes cadD/cadX (cadD; BX571858.1, pSAS [8203:8231] and cadX; BX571858.1, pSAS [8797:8827]).

CC130-MRSA-XI was identified in two Swedish hedgehogs; details have been reported separately [39]. Among the German samples, this strain was detected once each in a hedgehog, a red fox and a fallow deer as well as in two hares [104]. The isolates harbored mecC, the SCCmec XI-associated beta-lactamase gene and an arsenic resistance operon (arsB; FR823292.1, [29339:29366:r] and arsC; FR823292.1, [28080:28103:r]; [129]); they lacked lukF-P83/lukM but carried edinB and etD2 [39].

CC130-MSSA appears to be a common lineage in sheep and a major cause of infections in these animals (see [154], where it is named CC700), but we are not aware of observations in humans. Previously, CC130-MRSA-XI have been found in humans as well as in a wider variety of wild and domestic animals (see references quoted in the Introduction). Although there are no systematic studies yet available for most parts of the world, they seem largely to be geographically restricted to Western Europe. CC130-MRSA-XI in humans is rather rare. In Germany, about one out of 1,000 MRSA isolates belong to that strain [91]. In Denmark, it accounts for 2.7% of human MRSA infections. A zoonotic link of CC130-MRSA-XI appears to be possible [94] and domestic animals such as small ruminants might serve as hosts.

Clonal complex 133

This strain was found in a wild boar, roe deer, chamois and a mute swan. The isolates did not carry any resistance markers. Isolates also lacked tst1, enterotoxin genes sec/sel and lukF-P83/lukM although these genes are known to frequently occur in CC133 (see genome sequence of ED133, GenBank CP001996 and [2,155]). CC133-MSSA were previously detected in wild boar [19] and in Griffon vultures [12]. This strain was also frequently observed in small domestic ruminants, i.e., goats and sheep [2,3,154], but also in domestic pigs [123], cats, dogs, [156], cattle [155] and donkeys [157]. It could have been transmitted from wild to domestic ruminants, or vice versa, and the few reported human cases [116] could be related to contact to animals or animal products.

Clonal complex 398

One CC398-MRSA-V isolate was detected in a hare. It carried blaZ, erm(B), tet(K) and tet(M) but lacked relevant enterotoxin genes, PVL and lukF-P83/lukM. CC398 is a livestock-associated lineage with MSSA frequently been detected in poultry. CC398-MRSA-V was first discovered in the Netherlands in 2006 [16,17,83,84,85] and subsequently spread across several livestock species and humans in most of Western Europe. It was also occasionally detected in wildlife [13].

Sequence type 425

ST425 was (with 29 isolates) the most common lineage observed in this study. This included several isolates from roe deer, three isolates from red deer, one isolate each from wild boar and red fox as well as three isolates from badgers. For one German badger, generalized ulcerations were recorded.

Two of three badger isolates (including the one with the ulcerations) as well as one roe deer isolate carried the enterotoxin E gene see. These isolates had a truncated hlb gene although other IEC genes were absent. The other isolates lacked any genes from beta-hemolysin converting phages, see or sea, and they had an un-truncated hlb. One roe deer isolate was positive for the beta-lactamase operon, but other resistance markers were not found. No mecC-positive isolate was identified.

ST425 is a lineage that has been found in wild and domestic animals. Previously it was noted in wild boar from Germany [19] and red deer from Spain [12] as well as in humans. Patterns of transmission might be assumed in which domestic and wild ruminants might get infected by ingesting carnivore excretions, or in which carnivores might be infected by scavenging or by ingesting, i.e., wild berries (a common behavior in foxes and badgers) that might be contaminated with ruminant feces. The recent emergence of ST425-MRSA-XI warrants further monitoring. Such strains were reported from cattle in the United Kingdom [86] as well as from wildlife (fallow deer, wild boar) and environmental samples from Spain [158].

Clonal complex 599

This strain was found once, in a road-killed hedgehog from Thuringia. It carried SCCmec XI including mecC, blaZ and arsenic resistance determinants. All but one of the previously described PCRs [87] for the characterization of SCCmec XI yielded products of the expected sizes. The PCR with primers arsCM10/0061-F1: 5'-GACCACTCTTTACCTGCT-3'/tnp1S2 R1: 5'-AGATCATGGAAAACCGATCA-3', however, failed to amplify. These findings suggest that i) the SCCmec XI element was identical to those of CC130-MRSA-XI isolates [39,87] and ii) that the adjacent genomic (non-SCC) region was different. Phenotypically, the isolate was penicillin-, oxacillin- and cefoxitin-resistant.

Additionally, cadD/cadX cadmium resistance genes were detected (cadD; BX571858.1, pSAS [8203:8231] and cadX; BX571858.1, pSAS [8797:8827]). Regarding virulence markers, the isolate tested positive for tst1, sec, sel, “seU2” (an enterotoxin gene ALWH01000034.1, positions 21908 to 22660), the gamma-hemolysin locus and lukD/E while IEC genes, cna and sasG were absent. This isolate was virtually identical to a human isolate from The Netherlands [96].

CC599-MRSA-XI have previously been detected in cattle [159] and in a domestic cat [160]. To the best of our knowledge, there are no descriptions of CC599-MRSA-XI in wildlife, and to date no reports of CC599-MSSA at all.

Clonal complex 692

Swedish isolates of this strain included three from common magpies as well as one each from a great tit, a green woodpecker, a golden and a white-tailed eagle as well as from a tawny owl. Additionally, one isolate from bullous eruption at the knee of a red kite from Germany was tested. Enterotoxin genes were not detected, and resistance genes were absent.

This lineage has, according to the MLST database, previously been observed in a domestic pigeon from Turkey and a chicken from Northern Ireland. A CC692 chicken isolate from South Korea was sequenced recently (K12S0375, GenBank: JYGF) but this was a MRSA. Generally, CC692 appears to be a bird-related lineage although it recently was observed in a grey seal (Halichoerus grypus) that may have preyed upon marine birds [48], in slaughterhouse workers [142] as well as in Australian wallabies [56].

Clonal complex 707

One isolate belonging to CC707 was identified in a Swedish reindeer. It did not carry any resistance genes or IEC genes, and the genes, tst1, sek, seq, ccrA/B2 and the kdp locus were absent although they are commonly found in CC707 isolates (Strain 21235 GenBank AFTQ and author´s unpublished observations). CC707 have, to the best of our knowledge, not been reported previously from animals.

Sequence type 890

One isolate of this strain was identified, from a vole fecal sample and a spa type t1773 (04-82-17-25-17) was determined. The isolate carried the blaZ gene. The MLST database shows one entry only (http://saureus.mlst.net/sql/fulldetails.asp?id=1709&send=33), an isolate from a human from France. Wildlife observations of ST890 appear not to have been reported previously.

Clonal complex 1956

One isolate of this lineage was identified from vole feces. MLST was performed identifying ST1959. The isolate lacked enterotoxin, and PVL genes, lukF-P83/lukM, IEC genes as well as any resistance markers. According to the MLST database, ST1959 has previously been found in a beaver (Castor fiber). The CC’s predicted founder, ST1956, was detected previously in a red squirrel and the related ST1960 was isolated from a human in Poland.

Sequence type 2425

This novel ST (6-158-6-2-7-26-5) was identified from two isolates from European brown hares from Sweden. For one of them, abscesses in multiple organs were recorded. Isolates lacked any resistance genes but harbored tst1, sec, sel and ORF CM14. No resistance genes were detected. There are no related entries in the MLST database and therefore, no CC assignment is possible.

Sequence type 2691

This was a novel singleton ST (6-79-12-2-7-13-153) identified in isolates from two Swedish moose. One suffered from a large abscess on the right lateral hock, accompanied by inflammation of the joint and loss of articular cartilage. The other one was found dead with pneumonia and sepsis. Isolates did not harbor any resistance genes. Enterotoxin and IEC were not detected.

Clonal complex 2767

A novel, previously undescribed, ST (102-146-6-18-7-50-2) was identified twice, from the liver of a lynx and from an eye of a reindeer, both from Sweden. It was submitted to the MLST database and assigned ST2279. One isolate (from reindeer) harbored protease genes splA and splB as well as lukD which the other one lacked. Resistance, toxin and IEC genes were not detected. Later entries to the MLST database include two related STs (ST2767; 102-146-6-18-7-50-48 and ST3212; 102-146-416-18-7-50-48) that also originated from wildlife (i.e., from wild boars from Spain and Italy). ST2767 is the predicted founder of this clonal complex, hence the designation as CC2767.

Sequence type 2963

This was a new singleton sequence type. Its MLST profile was 6-79-6-2-13-50-48. It was isolated from the same road-killed wildcat from North Rhine-Westphalia as mentioned above (see CC49). Enterotoxin, IEC and resistance genes were not detected.

The authors also acknowledge the Division of Wildlife Diseases (National Veterinary Institute, Uppsala, Sweden), R. Mattsson and L. Rangstrup-Christensen for the collection and analysis of samples and for the pathology data. Dr. W. Rudolph (IMMH Dresden) helped with MLST sequencing and the staff of the IMMH Dresden as well as I. Engelmann (Alere) provided technical assistance.

We acknowledge the InfectoGnostics Research Campus Jena and Prof. E. Jacobs (IMMH Dresden) for support.