Introduction

Staphylococcus aureus is an opportunistic pathogen commonly found as part of the normal human microbiota, especially of the oropharynx, nose and skin, although it can also colonize the intestine and the perineal region. Most studies that analyze the S. aureus fecal colonization are referred to hospitalized patients [1, 18, 19, 38, 49], with different percentages reported depending on the studies, and very few reports are focused on fecal samples of healthy individuals [12]. In addition, very scarce data do exist on the molecular characterization of fecal S. aureus isolates of healthy humans. It is known that S. aureus colonization can precede infection and that it can adapt to different body sites [45]. S. aureus can also acquire different resistance mechanisms, highlighting the acquisition of the mecA gene, given that it confers resistance to almost all available beta-lactams [22]. Methicillin-resistant S. aureus (MRSA) can affect both hospitalized patients as well as patients from the community (CA-MRSA). In the last years, a new MRSA lineage (namely ST398, which is associated with farm animals, especially pigs) [14, 16] has emerged. Some other S. aureus genetic lineages are also associated with animals, such as ST133, which is mainly related to ungulates and ruminants [15, 41]. There is an increasing interest in the analysis of the circulating S. aureus genetic lineages in different ecosystems in order to determine the flux of lineages among different niches and their potential evolution.

S. aureus is also important due to its capacity to produce a large variety of virulence factors that can exacerbate the clinical situation of staphylococcal infections [25]. Some of these toxins are Panton–Valentine leukocidin (PVL), toxic shock syndrome toxin 1 (TSST-1), different hemolysins, enterotoxins, exfoliatins, and other virulence factors which facilitate cellular invasion, bacterial growth, and reduction of immune system human cells [9, 25, 40, 47].

S. aureus can acquire genes that seem to be associated with the capacity of this microorganism to evade the human immune system. This set of genes is called immune evasion cluster (IEC). The IEC system is located on an 8-kb region at the conserved 3′ end of β-hemolysin-converting bacteriophages. This region contains the genes scn, chp, sak, and sea or sep, which have different functions to achieve the survival of S. aureus within our innate defenses. The presence of different combinations of these genes in S. aureus allows its classification into different IEC types [39, 47].

The aims of this study are to determine the rate of S. aureus fecal carriage in healthy humans and to analyze the genetic lineages, the resistance genes and virulence determinants of recovered isolates. Furthermore, we focused on the determination of the IEC types to hypothesize on the possible human or animal origin of isolates.

Materials and Methods

Bacterial Isolation

Fecal samples of 100 healthy human volunteers were obtained during September 2010 and March 2011 in La Rioja (northern Spain). Healthy humans (age range 2–89 years; 53 % women and 47 % men) had neither received antibiotics nor had relation with the hospital environment for at least 3 months prior to sampling, and all gave their informed consent (or their parents in case of underage) to participate in this study. Direct suspension of the fecal swabs was performed in sterile saline solution, and 100 μL was inoculated into brain–heart infusion broth containing 6.5 % NaCl and incubated at 37 °C for 24 h. Then, 100 μL of this culture was seeded on mannitol–salt agar (MSA, Beckton–Dickinson), and oxacillin resistance screening agar base (ORSAB), OXOID) supplemented with 2 mg/L of oxacillin, and the plates were incubated at 37 °C for 24–36 h. Presumptive S. aureus and MRSA colonies were selected in MSA (yellow colonies) and ORSAB plates (blue colonies), respectively (two per positive plate). Identification of S. aureus and MRSA was performed by conventional methods (DNAse assay, catalase, and Gram staining) and confirmed by specific duplex PCR of the nuc (S. aureus specific thermonuclease) and mecA (staphylococci methicillin-resistance determinant) genes [30]. S. aureus isolates were stored at −80 °C until further analysis. All isolates were tested for their capacity to coagulate the bovine plasma (Sigma–Aldrich) following standard methodology [20].

Molecular Typing of Isolates

S. aureus isolates were characterized by different molecular methods. Single-locus DNA sequencing of the hyper variable region of S. aureus protein A (spa) [21] was performed, and the obtained sequences were analyzed using the Ridom StaphType software version 1.5.21 (Ridom GmbH). Detection of agr allotypes—accessory gene regulatory loci—was carried out by two multiplex PCR [43] in all isolates. Multi-locus sequence typing (MLST) was also performed in all S. aureus isolates to determine the sequence type (ST) and the clonal complex (CC) of each isolate (www.saureus.mlst.net).

Susceptibility Testing and Detection of Antimicrobial Resistance Genes

Susceptibility testing was carried out by disk-diffusion agar method for 17 antimicrobials (penicillin, oxacillin, cefoxitin, erythromycin, clindamycin, ciprofloxacin, gentamicin, streptomycin, kanamycin, tobramycin, tetracycline, chloramphenicol, trimethoprim–sulfamethoxazole, linezolid, vancomycin, mupirocin, and fusidic acid) following the CLSI recommendations [7, 8], except for fusidic acid, mupirocin, and streptomycin for which SFM guidelines were followed [6]. In addition, the D-test was performed for the detection of inducible clindamycin resistance [7]. The presence of the erm(A), erm(B), erm(C), erm(T), msr(A), msr(B), mph(C), ant(4′)-Ia, mup(A), and blaZ resistance genes was studied by PCR (and sequencing for some of the amplicons) [17, 30, 42, 44].

Detection of Virulence Genes

The presence of the genetic determinants of the Panton–Valentine leukocidin (lukF/lukS-PV), toxic shock syndrome toxin (tsst-1), exfoliative toxin A (eta), B (etb), and D (etd), as well as alpha-(hla), beta-(hlb), delta-(hld), gamma (hlg), and gamma-hemolysin variant (hlgv) was investigated by PCR. In addition, the genes encoding leukocidins DE (lukD-lukE) and M (lukM), aureolysin (aur), the biofilm-associated protein bap, and the adhesion factor cna were also studied by PCR [9, 25, 28, 30, 31, 40, 50, 51]. Eighteen staphylococcal enterotoxin genes (SEs) (sea, seb, sec, sed, see, seg, seh, sei, sej, sek, sel, sem, sen, seo, sep, seq, ser, and seu) were analyzed by PCR and PCR multiplex [23].

Immune Evasion Cluster

The five genes of the IEC cluster (scn, chp, sak, and sea or sep) were studied by PCR and sequencing; according to their presence or absence, S. aureus isolates were classified into seven IEC types following previously described patterns [47].

Results

Fecal Carriage and Molecular Typing of S. aureus

Fifteen of the 100 tested fecal samples (15 %) were positive for S. aureus on MSA plates. As both isolates of each positive sample showed similar resistance phenotype and spa type, only one isolate was maintained and further studied. All 15 isolates were susceptible to oxacillin and cefoxitin, and were negative for mecA gene; therefore, they were MSSA. No MRSA isolates were recovered from ORSAB medium.

Complete characterization of the 15 MSSA isolates is shown in Table 1. A high diversity of spa types was detected (t002, t012, t021, t084, t136, t166, t209, t216, t571, and t3495), including a new spa type registered as t7866 in Ridom SpaServer. The detected agr allotypes were I, II, and III, grouping all isolates. The S. aureus isolates belonged to eight different sequence types that were included into seven different clonal complexes, highlighting the presence of strains typed as ST398 (spa-t571), ST133 (spa-t3495), and ST5 (spa-t002) ascribed to the clonal complexes CC398, CC133, and CC5, respectively. Other sequence types identified were (ST(spa type)/CC ascribed): ST30(t012 or t021)/CC30, ST34(t136, t7866 or t166)/CC30, ST59(t216)/CC59, ST15(t084)/CC15, and ST109(t209)/CC9. Forty percent of the strains were included within clonal complex CC30. These strains fell into two sequence types, ST30 and ST34. MSSA isolates lacked the ability to coagulate bovine plasma except the isolate typed as ST133(t3495)/CC133.

Table 1 Characteristics of the 15 MSSA isolates from fecal samples of healthy humans recovered in this study
Full size table

Susceptibility Testing and Detection of Antimicrobial Resistance Genes

Few antimicrobial resistance traits were identified among our strains. Nine of them showed penicillin resistance (carrying the blaZ gene), three strains erythromycin resistance (carrying the erm(A), erm(C), erm(T) or mph(C) genes], and one strain exhibited tobramycin and mupirocin resistance (carrying ant(4′)-Ia and mup(A) genes). Four strains were susceptible to all tested antimicrobial agents (Table 1).

Virulence Genes Detected

A high diversity of virulence genes was identified among the 15 S. aureus strains (Table 1). Interestingly, six strains contained the tsst-1 toxin gene (40 %) and one strain harbored the exfoliative toxin A gene (eta). All MSSA strains carried the hla and hld genes. Other hemolysin genes detected were as follows (number of strains): hlb (nine), hlg (six), and hlgv (nine). The lukD-lukE genes were detected in five strains, and the lukM gene was identified in the strain CC133. All strains except the one ascribed to CC133 carried the aur gene, while only two strains (both belonging to CC30) were positive to cna gene. All strains were negative for the presence of the genes lukF/lukS-PV and bap. None of our strains harbored the enterotoxin gene cluster named egc (seg, sei, sem, sen, and seo); however, 20 % presented the enterotoxin genes seg, sei, sem, sen, seo, and seu, included in the enterotoxin gene cluster like (egc-like) operon [4, 24]. All S. aureus strains lacked see and ser enterotoxin genes (Table 1).

Analysis of Immune Evasion Cluster Genes

All S. aureus strains except two harbored the genes of the IEC system. Only the strain of lineage CC133 and one ascribed to CC5 did not present the IEC genes. The following IEC types were obtained in the remaining 13 MSSA (IEC type/ST–CC): IEC type A/ST30–CC30; IEC type B/ST34–CC30, ST59–CC59, or ST109–CC9; IEC type C/ST15–CC15, ST59–CC59 or ST398–CC398; IEC type E/ST34–CC30; IEC type F/ST5–CC5; and IEC type G/ST34–CC30. Six strains were ascribed to the IEC type A/ST30-CC30, IEC type B/ST34-CC30 and IEC type C/ST15-CC15. The IEC type D was not detected among our strains (Table 1).

Discussion

A moderate rate of S. aureus fecal carriage (15 %) was detected among the healthy humans in this study. Few reports of this type have been previously performed, and most of them were focused on intestinal S. aureus carriage in hospitalized patients, with occurrences ranging from 8 to 31 % [18, 19, 49]. There are very few studies on healthy individuals, and as far as we know, none on genetic lineages of S. aureus from the intestinal ecosystem of healthy people.

The few available studies that analyzed the prevalence of intestinal colonization by S. aureus found values ranging from 6 % in healthy children [12] to 20 % in people with various clinical situations [1]. The absence of MRSA detection in our study is of relevance, although low percentage of MRSA carriage rates (0–5 %) has been reported in previous studies in healthy children [12], or in people at the time of hospital admission [3, 13, 26]. However, a higher prevalence rate was detected among hospitalized patients in other study (24 %) [27]. One question would be if S. aureus is a passenger or a real colonizer of the intestinal tract. A previous study evaluated the frequency of nasal and extranasal MRSA carriage on patients admitted at a hospital, identifying a strong association of extranasal carriage with nasal MRSA colonization [3].

The high genetic diversity of spa types detected among our strains (11 different spa types, one of them new) is remarkable. Most of the spa types identified were related to the clonal complex CC30, a lineage that seems to have become a successful colonizer of humans [2, 35] and is also considered one of the most frequent S. aureus lineages detected in human infections [45]. The detection of a MSSA strain of the sequence type ST398, which is traditionally associated with farm animals (especially pigs), and a MSSA strain belonging to sequence type ST133, related to ungulates and ruminants, suggests the incipient ability of this microorganism to colonize different animal or human hosts away from their usual niches [15, 31]. In addition, as far as we know, S. aureus of lineage ST133 has not previously detected in healthy humans. Some authors have hypothesized about the ancestral human origin of genetic line CC133 and the possible evolutionary leap to ruminants many centuries ago [20]. Moreover, recent studies have suggested that the CC398 lineage could be originated in humans as MSSA and then spread to livestock, where it subsequently acquired the mecA gene and became well adapted to these animals [36]. It is of interest to indicate that the healthy individual who carried the ST133 isolate worked in a food–animal farm, but the one with ST398 had no relation with farm animals. The two healthy individuals who carried MSSA of the lineages ST398 and ST133 were negative for S. aureus nasal carriage (data not shown). We cannot discard the implication of the food chain in the acquisition of these S. aureus clones.

The presence of the IEC system, especially of the scn gene, could indicate the possible human origin of a S. aureus strain, encoding staphylococcal complement inhibitor (SCIN) [39]. SCIN is a staphylococcal inhibitor of human complement system, in fact, is a C3 convertase inhibitor, blocking the formation of C3b on the surface of the bacterium and the ability of human neutrophils to phagocyte S. aureus. In our case, the presence of the genes of IEC system and the absence of mecA gene in the detected strain ST398/CC398 indicate that it might belong to the human-associated subclone. By contrast, given that the strain typed as ST133/CC133 does not contain the genes of IEC system and presents capacity to coagulate bovine plasma, it is estimated to belong to the well-adapted ungulates and ruminants lineage. Moreover, the detection of strains of lineage ST5 with and without IEC genes is also of interest. According to some authors, S. aureus strains of ST5 lineage could have evolved in time jumping from different hosts and acquiring different adaptability mechanisms [29]. It is interesting to underline the wide variety of IEC types detected among our strains CC30 (A, B, E, and G).

All our S. aureus strains were PVL-negative. The lukF/lukS-PV genes encoding the Panton–Valentine leukocidin are located in phages, and it is believed that these genes could be transferred from MSSA into CA-MRSA [10]. A high proportion of S. aureus strains (40 %) carried the tsst-1 toxin gene, most of them of the clonal complex CC30. Similar results have been found in other studies of nasal carriers (close to 30 %) [30, 34]. TSST-1 is a toxin related to the toxic shock syndrome. The association between TSST-1 and CC30 has been previously described [10]. One strain (6 %) ascribed to CC9 carried the eta gene, encoding the exfoliative toxin A, associated with the staphylococcal scalded skin syndrome and impetigo [33]. Previous studies remark a high association between the clonal complex CC9 and its ability to produce exfoliative toxins [2].

All strains were positive for hla and hld genes, and high percentage of them were also positive for other hemolysin genes (hlb, hlg, and hlgv). These toxins are produced by a large amount of S. aureus strains [11] and have the ability to destroy red blood cells, especially when S. aureus reaches the blood and causes bacteremia. One-third of the strains harbored the lukD-lukE genes, and only the strain CC133 contained the lukM gene encoding a leukocidin, prevalent in isolates of ungulates and ruminants [15, 41].

All MSSA strains except the one of the lineage ST133 carried the aur gene encoding the metalloproteinase aureolysin. This fact points again to the animal origin of our strain ST133, due to the capacity of this enzyme to activate prothrombin in human plasma [37, 40]. The absence of the cna gene and the tetracycline-susceptible phenotype in strain CC398 might be also a good marker of human origin, given that presence of the cna gene and tetracycline resistance are frequently detected in livestock-associated MRSA CC398 strains [32]. Moreover, none of the S. aureus strains harbored the biofilm-associated bap gene. It has been previously suggested that the prevalence of the gene bap in S. aureus strains is very low [9, 48].

Only three strains harbored the egc-like operon and belonged to ST109/CC9 and ST30/CC30. This cluster is normally found on strains of these clonal complexes [2]. The remaining 12 strains were positive at least for one SE gene; 2 of them belonged to ST34/CC30 and harbored incomplete egc-like cluster, lacking the seo gene. Incomplete egc or egc-like clusters have been detected in previous studies [5].

Regarding the antimicrobial resistance pattern of investigated strains, only 60 % showed resistance to penicillin and harbored the blaZ gene. Higher rates are normally detected among the nasal S. aureus population of healthy individuals [30]. In addition, three strains were erythromycin- and clindamycin-resistant and presented the resistance genes erm(A), erm(C), erm(T) or mph(C); these strains belonged to CC9, CC15, and CC398 lineages. The resistance profile of human-associated CC398 lineage frequently includes resistance to macrolides and lincomycins and the presence of erm(T) gene [17, 31, 46], similar to our case. One strain belonging to CC30 harbored the ant(4′)-Ia and mupA genes encoding resistance to tobramycin and mupirocin, respectively. It is important to remark the detection of a mupirocin-resistant strain in the human intestinal microbiota because this antibiotic is the treatment of choice to decolonize the human nasopharynx [30]. The narrow antimicrobial resistant phenotype and genotype found among the studied strains fall within the normal low range typically identified on MSSA strains.

A moderate rate of S. aureus fecal carriage has been detected in this study (15 %) with a wide variety of genetic lineages identified. The detection of MSSA strains of the lineages ST398(t571)/CC398 and ST133(t3495)/CC133 is of relevance. Data from our study suggest that strain ST398(t571)/CC398 belongs to the subclone associated with humans, given that it preserves the genes of IEC system, remains negative for the mecA gene, and is tetracycline-susceptible. Nevertheless, more studies should be performed in the future to analyze the genetic lineages of big collections of MSSA of healthy humans and animals to deepen the knowledge of the origin and evolution of this microorganism in different niches.