spa Types and Staphylococcal Enterotoxin Production of Staphylococcus aureus Isolated from Wild Boar

Little is known about the structure of S. aureus population and the enterotoxin gene content in wild boar. In 1025 nasal swabs from wild boars, 121 S. aureus isolates were identified. Staphylococcal enterotoxin (SE) genes were identified in 18 isolates (14.9%). The seb gene was found in 2 S. aureus isolates, sec in 2 isolates, the see and seh genes were found in 4 and 11 isolates, respectively. The production of SEs was evaluated in bacteria grown in microbial broth. Concentration of SEB reached 2.70 µg/ml after 24 h and 4.46 µg/ml at 48 h. SEC was produced at 952.6 ng/ml after 24 h and 7.2 µg/ml at 48 h. SEE reached 124.1 ng/ml after 24 h and 191.6 ng/ml at 48 h of culture. SEH production reached 4.36 µg/ml at 24 h and 5.42 µg/ml at 48 h of culture. Thirty-nine spa types were identified among S. aureus isolates. The most prevalent spa types were t091 and t1181, followed by t4735 and t742, t3380 and t127. Twelve new spa types, i.e., t20572‒t20583 were identified. The wild boar S. aureus population was shown to contain previously identified animal/human-associated spa types and spa types not identified in humans or animals. We also indicate that wildlife animals can be a significant reservoir of see-positive S. aureus. Supplementary Information The online version contains supplementary material available at 10.1007/s00248-023-02236-4.


Introduction
Staphylococcus aureus is considered one of the most common opportunistic pathogens of humans and animals. This bacterial species is responsible for a wide range of infections, from superficial skin and tissue infections to serious, life-threatening diseases such as pneumonia and sepsis [1]. Furthermore, staphylococcal enterotoxins (SEs) produced by many staphylococcal strains are a cause of staphylococcal food poisoning (SFP) which makes S. aureus an important foodborne pathogen [2].
S. aureus can colonise both animals and humans. Due to changes in the natural environment, previously isolated ecological niches are now frequently overlapping [3]. It was shown that specific staphylococcal lineages have passed from one host to another and that humans can act as a source of pathogenic strains of S. aureus that affect animals. S. aureus belonging to clonal complex 5 (CC5) was shown to pass from human to poultry [4] and CC398 from human to pig [5] as well as other livestock and companion animals [6,7]. The adaptation of S. aureus to a new niche changes many of its properties, including those involved in pathogenesis. For a number of clonal lineages host specificity is less pronounced and therefore adaptation to new hosts is more frequent (e.g. ST130, CC398) [8]. Therefore, animals can serve as a source of new altered pathogens for humans [9].
Very little is known about the occurrence and genetic diversity of S. aureus in the wild boar population. Meemken et al. [10] concluded that S. aureus is a rare coloniser of wild boars, as this pathogen was isolated from 6.8% of the investigated animals. Among these isolates, spa types t1181, t6782, and t6384-t6386 were identified. According to Porrero et al. [11] 17.6% of the wild boar population carry S. aureus. The isolates belonged to 36 spa types, the most common of which was t3750. In these studies, no CC398 S. aureus isolates were found, suggesting a low frequency of this clonal complex in wild boars. However, in 2017 the first isolation of CC398 from a wild boar was reported [12].
Ramos et al. [13] obtained 57 isolates of S. aureus from 177 nasal swabs from wild boars from Portugal. The most prevalent spa type was t11502 with 37 isolates, and the presence of t011 and t034 isolates that are typically associated with livestock was found in 9 and 8 isolates, respectively.
Even less is known about the enterotoxigenic potential of wild animal-derived S. aureus. In a study by Seinige et al. [14], 36.9% S. aureus cultures were found in 111 wild boar nasal samples. The researchers screened the isolates for the presence of staphylococcal enterotoxin genes (sea-see and seh). The only enterotoxin gene found was seh. It was present in 11 isolates, 5 of which belonged to the t127 spa type.
In this study, spa typing was used to investigate the genetic diversity of S. aureus isolates obtained from wild boars in Poland. The enterotoxigenic potential of wild boar S. aureus isolates was investigated. S. aureus was tested for the sea-see and seh genes encoding SEs, which are considered the most important food safety hazards.

Isolation and Identification of S. aureus
One thousand twenty-five nasal swabs were taken from wild boars (Sus scrofa) at a game collection point in north-west Poland during the years 2014-2017. The swabs were taken by placing a sterile cotton swab approximately 10 cm into the nares. Individual swabs were placed in 20 ml of Giolitti-Cantoni broth (Merck, Germany) and cultured statically for 24-48 h at 37 °C. Subsequently, cultures were streaked on Baird-Parker agar (Merck, Germany) for further selection of staphylococcal isolates and incubated for 24-48 h at 37 °C. Single colonies displaying the characteristic morphology of S. aureus were transferred to 5 ml of BHI broth (BTL, Poland) and cultured at 37 °C with agitation for 18-24 h. Species identification was confirmed by PCR, using primers for the nuc and clf genes, encoding thermonuclease and clumping factor, respectively, as previously described [15]. One S. aureus isolate per sample was taken for further analysis. The isolates were stored in BHI medium with 15% glycerol.

Genomic DNA Extraction
Two-millilitre aliquots of an overnight bacterial culture in BHI broth (BTL, Poland) were centrifuged at 12,000 × g for 5 min. The bacterial pellet was resuspended in 150 μl 0.1 M Tris-HCl buffer, pH 7.4, containing 2 units of lysostaphin (A&A Biotechnology, Poland), and incubated at 37 °C for 30 min. Then 15 µl of 10% SDS was added and incubated at 37 °C for 10 min, followed by the addition of 200 µl of 5 M guanidine hydrochloride solution and incubation for 10 min at room temperature. DNA was extracted by phenol and chloroform, precipitated by ethanol, dissolved in 50 μl of UltraPure™ Distilled Water (Thermo Fischer Scientific Inc., USA) and stored at − 20 °C.

Detection of Staphylococcal Enterotoxin Genes
S. aureus genomic DNA was screened for the sea-see and seh genes encoding the SEs being the most important food safety hazards. The detection of staphylococcal enterotoxin genes sea-see was performed according to Sharma et al. [16]. Detection of the seh gene was performed as described previously [15].

S. aureus spa Typing
For all S. aureus isolates, spa types were determined using the method described by Harmsen et al. [17]. PCR amplicons were sequenced (Genomed, Poland). For spa typing and cluster analysis of spa types with the minimum spanning tree algorithm, Ridom SeqSphere + software was used [18]. spa types shorter than 5 repeats were excluded from the analysis. Where possible, isolates were assigned to MLST clonal complexes (CCs), based on information available in the PubMLST database (https:// pubml st. org/) and literature resources.

Bacterial Growth Conditions and Sandwich ELISA
For selected enterotoxigenic isolates representing different spa types, a growth curve and enterotoxins production using ELISA were determined. Isolates in which enterotoxin genes were identified were subtyped using RAPD-PCR according to Louws et al. [19], then within enterotoxigenic isolates representing a given spa type, all isolates with different RAPD profiles were selected for ELISA. A single colony was inoculated in 5 ml of BHI and incubated overnight at 37 °C. Subsequently, the cultures were diluted to OD 600 = 0.03 in 5 ml of BHI broth and incubated for 48 h with cell count and ELISA assay after 24 and 48 h. Bacterial cells were quantified by plating serial dilutions of the culture on BHI agar. Sandwich ELISA for SEC and SEH was performed as previously described [15], and recombinant SEC and SEH produced as previously described [15] served as controls. The antiserum for the detection of SEB was purchased from Acris (Herford, Germany), and the antiserum for SEE was purchased from Abcam (Cambridge, UK). Sandwich ELISA for SEB and SEE was performed as previously described [15]. Purified SEB (Sigma-Aldrich, USA) and recombinant SEE (Abcam, UK) served as controls. The concentration of enterotoxins in samples was determined using a four-parameter logistic curve. Data analysis was carried out using GraphPad Prism software (GraphPad Software Inc., USA).

Identification of S. aureus in Wild Boars and Staphylococcal Enterotoxin Genes Content
In 1025 nasal swabs from wild boars, 121 S. aureus isolates were identified. Genes of at least one SE were found in 16 isolates (13.2%). The seb gene was found in 2 S. aureus isolates (1.7%), sec in 2 isolates (1.7%), see and seh gene were found in 4 (3.3%) and 11 (9%) isolates, respectively. One S. aureus isolate was found to carry both see and seh genes (isolate 35WB), and two isolates carried both sec and see genes (isolates 92WB, and 101WB) (Supplementary Table S1).

Growth and Production of Staphylococcal Enterotoxins by S. aureus Isolates from Wild Boars
In 10 isolates in which SE genes were found by PCR, the bacterial number and staphylococcal enterotoxins production (SEB, SEC, SEE and SEH) was tested using ELISA at 24 and 48 h of culture. The numbers of bacteria ranged from 9.5 ± 0.2 to 9.9 ± 0.2 log CFU/ml after 24 h of culture and from 9.8 ± 0. 4

Genotypes of S. aureus Isolates from Wild Boars
Thirty-nine unique spa types were identified among 121 S. aureus isolates ( Table 2). The most prevalent spa types were t091 and t1181, with 18 isolates each, followed by t4735 with 16 isolates. The spa types t742, t3380 and t127 were represented by 9, 7 and 5 isolates, respectively. Sixteen S. aureus isolates were assigned to 12 new spa types (t20572-t20583). Based on spa type similarity, the isolates were assigned to 5 spa clusters (Fig. 1). Similarity could not be calculated for spa types that contained less than 5 repeats, i.e., t3369, t3424, t3625, t639, and t9909. Eighty-one out of 121 S. aureus isolates were assigned to MLST clonal complexes (CCs) or sequence types (ST) based on literature and PubMLST database. Largest spa cluster 1 was formed by 44 S. aureus isolates. Thirty-four of them were assigned to clonal complex CC133 (Fig. 1). spa cluster 2 was formed by 19 isolates. This cluster contained 9 isolates assigned to CC425 as well as 6 out of 12 new spa types identified in this study. From 3 isolates forming spa cluster 3, one was assigned to CC101. Six out of 8 isolates forming spa cluster 4 were assigned to CC1. spa cluster 5 consisted of 3 isolates, 2 of which were assigned to CC133. All other S. aureus were assigned as singletons based on spa type similarity. From this, 18 isolates were assigned to CC7, 3 isolates to CC30, 3 isolates to CC97, 3 isolates to CC5, one isolate to CC398 and one isolate was assigned to ST2328 (Fig. 1).

Discussion
S. aureus is a ubiquitous coloniser of both humans and animals. Numerous studies have indicated host-specificity of certain clonal lineages of this bacterial species [9]. However, host adaptation of S. aureus does not exclude incidental jumps of certain clones to other hosts. It has been shown that livestock-associated S. aureus strains may colonise and cause zoonotic infections in humans. In turn, certain human S. aureus clones have been shown to pass to animals where they adapt to the new host [8].
Nine of the bacterial isolates studied here belonged to spa type t742. All of these isolates were included in spa cluster 2. The t742 genotype, assigned to CC425 was identified in wild boars [14] and recently in surface waters [31]. Based on the number of hosts in which a given CC was found and the number of isolates identified within the CC, the S. aureus clonal complexes were divided into host generalist and host specialist [8]. According to this assumption, CC425 was classified as a host specialist. Human is not a common host for S. aureus CC425 [8].
We identified six wild boar S. aureus isolates belonging to CC1. The occurrence of this clonal complex in livestock was already observed [26,[32][33][34], the isolation of these clones has been also reported in humans [25,35]. Three wild boar S. aureus isolates were assigned to CC5. Previous observations by Lowder et al. [4] indicate that most S. aureus isolates from diseased and healthy poultry from Belgium, China, Denmark, Japan, the UK, and the USA belonged to the CC5 complex [26,29,36]. One wild boar S. aureus was assigned to t056 belonging to ST101. This clonal complex is mostly associated with humans [37], but it has also been isolated from cattle [38], non-human primates [37], and rabbits [39]. We identified three wild boar S. aureus isolates as members of CC97. This complex is believed to be associated with human and livestock, and S. aureus belonging to CC97 was identified in cattle and pigs [40]. The wild boar S. aureus population contains previously identified animal/ human-associated genotypes, and genotypes not identified in humans or animals.
Most of the enterotoxins identified so far have emetic activity, as demonstrated in experiments conducted in animal models [2]. However, involvement in SFP cases was only demonstrated for a part of known SEs [41]. For this, the screening of wild boar S. aureus was focused on Fig. 1 Clustering of spa types of wild boar S. aureus isolates. Enterotoxigenic isolates were identified using PCR for sea-see and seh genes. Calculation of similarity between spa types and construction of minimum spanning tree was performed using Ridom SeqSphere + . Similarity could not be calculated for spa types that contained less than 5 repeats, i.e., t3369, t3424, t3625, t639, and t9909 SEA-SEE and SEH, enterotoxins whose involvement in SFP was widely confirmed [42,43]. The see gene was identified in an S. aureus isolate belonging to spa cluster 4 and assigned to CC1, in one isolate belonging to spa cluster 2 and in two isolates closely related to this cluster. Two S. aureus isolates harbouring the see gene were assigned to new spa type, i.e., t20575. The see-positive S. aureus isolate t4279 was assigned to spa cluster 2 in which we identified a spa type (t742) belonging to CC425, and two isolates of t20575 are closely related to spa cluster 2. In turn, one see-positive isolate was assigned to t922 spa type, which is closely related to t127. t922 was already identified as human, community-associated S. aureus [44]. So far, little is known about the occurrence of see in S. aureus. S. aureus carrying this gene was already detected in cows [45], and was listed as the cause of an SFP outbreak in France [46]. Recent research based on whole genome analysis demonstrated that within 883 isolates of S. aureus, mainly from human clinical cases and livestock animals, no see gene was identified [47]. However, in a recently published whole genome sequence, the see gene was identified in S. aureus from a wildlife isolate, i.e., European badger (GenBank: CP097571.1). Based on the CP097571.1 genome analysis, we identified the ST of this isolate as closely related to ST425. As mentioned above, this ST belongs to CC425, a host specialist complex that mainly includes animal S. aureus isolates. Evidence of an SFP outbreak due to SEE comes from France, 2009 [46]. In this investigation, the total intake of SEE per body was estimated to be 90 ng. Furthermore, the authors quantified SEE in the food sample to be 0.36 to 1.1 ng/g. Our isolates produced SEE at 80-124 ng/ml at 24 h and 102-192 ng/ ml at 48 h of culture in microbial broth.
Other SEs produced by S. aureus from wild boar include SEB, SEC, and SEH. The seb gene was identified in two S. aureus isolates assigned to the spa type t160. This spa has already been found in human clinical cases [48], but also in livestock such as chickens and rabbits [49,50]. The sec gene was identified in two S. aureus isolates simultaneously carrying the see gene. The seh gene was found in isolates belonging to CC1 and CC133. Some of the SEs investigated were produced at concentrations exceeding a few micrograms per millilitre of medium. In particular, SEB production reached 2.6 and 4.5 µg/ml at 48 h of culture.
Production of enterotoxins by wild boar S. aureus isolates was not assessed previously. We identified wild boar S. aureus isolates which carried and expressed the enterotoxin genes seb, sec, see, and seh. The see gene rarely found in already characterised populations of S. aureus was identified in three isolates displaying unrelated spa types. Our results suggest that wild boar can be a significant reservoir of seepositive S. aureus.
Author Contribution S.B. and J.Ba. designed the study and wrote the manuscript. S.B., A.T. and B.W. performed the experiments. K.Ł. and J.By. obtained the bacterial isolates. All authors reviewed the manuscript.
Funding This study was partially funded by the Polish National Agency for Academic Exchange under Grant No. PPI/ APM/2019/1/00044/U/00001.

Declarations
Ethics Approval This article does not contain any studies with human participants or animals performed by any of the authors.

Conflict of Interest
The authors declare no competing interests.
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