Staphylococcus sp. are one of the most commonly found pathogenic bacteria in human environment. The epidemiology of Staphylococcus species in animals has gained interest in the last years, not only for their importance in veterinary medicine, but for its increasingly evidenced zoonotic potential.  The  genus  includes  both human and animal pathogens, generally coagulase-positive staphylococci (CPS) such as S. aureus, S. intermedius, S. delphini, S. hyicus, S. schleiferi subsp. coagulans, and S. pseudintermedius, and coagulase negative staphylococci (CNS) such as S. equorum, S. xylosus, S. saprophyticus, S. succinus, S. warneri, S. epidermidis, and S. lentus (Devriese et al., 2008). Staphylococcus aureus is a dangerous human pathogen responsible for a wide variety of diseases. Other species are difficult to identify as frank human pathogens, but a few reports have described other coagulase positive staphylococci as causing opportunistic infections (Van Hoovels et al., 2006).

Nearly all S. aureus strains secrete exoproteins such as coagulase, nucleases, proteases, lipases, hyaluronidase and collagenase. Staphylocoagulase (SC) causes coagulation of plasma and is regarded as a marker for discriminating S. aureus from other less pathogenetic staphylococci. The nuclease enzyme is the major regulator of S. aureus virulence determinants (Olson et al., 2013) and the amplification of the nuc gene has a potential for the rapid diagnosis of S. aureus infections (Costa et al., 2005). Staphylococci secrete a wide spectrum of diverse extracellular proteins that are coordinately expressed during different stages of infection by a network of virulence regulators which render the bacterium virulent (Cotar et al., 2010). These include adhesion factors, toxic proteins/enzymes, and exotoxins including exfoliative (eta, and etb), staphylococcal enterotoxins (SEs), and toxic shock syndrome toxin-1 (TSST-1) (Nemati et al., 2013).

As major virulence factors of S. aureus, TSST-1, and ETs (A and B) are pyrogenic toxins that have been implicated in host colonization, invasion of damaged skin and mucus, and evasion of host defense mechanisms. They are responsible for specific acute staphylococcal toxemia syndromes (Udo et al., 2009). Exfoliative toxins (also known as “epidermolytic” toxins) are particularly interesting virulence factors of S. aureus. These extremely specific serine proteases recognize and cleave desmosomal cadherins only in the superficial layers of the skin, which is directly responsible for the clinical manifestation of staphylococcal scalded skin syndrome (SSSS) (Bukowski et al., 2010). However, a significant increasing rate of ETs was recorded for nasal and clinical isolates (DaÄŸi et al., 2015). The production of eta and etb has been examined in S. aureus strains and other strains associated with SSSS (Plano, 2004). Other CPS species, such as S. intermedius and S. hyicus, produce similar toxins (Ahrens and Andresen, 2004). However, it has not been fully understood whether the toxins are produced only by human strains or whether animal strains also produce   them.  Direct   contact   between animals   and humans is a relevant factor to take into account to understand the prevalence and the evolution of Staphylococcus species. As S. aureus could pass from livestock to humans, it could be public health problem.

Clinically, toxic shock syndrome is closely associated with strains of S. aureus carrying the gene encoding for tst and associated mostly with tampon use (McDermott and Sheridan, 2015). The gene encoding tst is a chromosomal, and the toxin is symptomatically related to the staphylococcal enterotoxin group of toxins which are included in the pyrogenic toxin superantigen families (PTSAgs). PTSAgs exert their virulence by binding to the major histocompatibility complex (MHC) class II molecules and the Vβ chain of the T-cell receptor (TCR) from the outside in a nonspecific manner. This leads to the stimulation of T-cell proliferation, the release of inflammatory cytokines, and ultimately the suppression of the host immune system (Larkin et al., 2009).

Due to the high sensitivity and specificity they provide, molecular markers are an alternative tool for accurate identification and classification of Staphylococcus species. Molecular assays targeting some genes such as hsp60, 16S rRNA gene, femA, dnaJ (Hauschild and Stepanovic, 2008), and catalase (kat) gene have been used for reliably identifying and classifying staphylococci (Blaiotta et al., 2010). The aim of the present study was to characterize CPS isolated from human and animal sources in Jordan and to investigate the prevalence of ETs and tst genes among the isolates. In addition, PCR-RFLP analysis of the kat gene was employed for a genotypic study. 

Collection of samples

A total of 753 samples were collected from human and animal sources during the period from October 2012 to May 2013.

Human sources

One hundred healthy students (53 female and 47 male) at Al-Balqa' Applied University were enrolled as volunteers. Two swabs were obtained from each student, one from nasal, and the other from nail. A written informed consent was obtained from all the volunteers in the study. Swabs were incubated on Tryptic Soy Broth (TSB) supplemented with 7% NaCl for 24 to 48 h at 37°C. In addition, 73 S. aureus isolates from routine microbiological specimen were collected from different hospitals in Jordan. The isolates were obtained from cultures of different specimens including blood infection, urinary tract infection, abscess, and septicemia. Suspected colonies of S. aureus from primary culture plates of Blood agar (BA), and Mannitol Salt Agar (MSA) were confirmed, by Gram reaction, positive catalase, tube coagulase and Deoxyribonucleases (DNAse) test. Nasal, nail, and clinical isolates were subcultured on MSA and Baird Parker Agar (BPA), and incubated at 37°C for 24 to 48 h. The suspected colonies were maintained on Staphylococcal agar no.110 (Fabiano et al., 2008) and were kept at 4°C for identification (Abdul Aziz et al., 2013).

Animal sources

Four hundred and eighty samples were collected from three central slaughterhouses in Jordan which represent the population of animals in Amman city (capital of Jordan). Samples were imported meat (Romanian, Australian) from177 sheep, 47 goats, and 16 cows. The age of the animals ranged from 1.5 to 3 years. Two samples were obtained from each animal, one from nares before slaughtering and others from various muscle sites by collection of segments of the muscle or from swabs of slaughtered animals. Samples were enriched in TSB containing 7% NaCl for 24 to 48 h of incubation at 37°C (Isenberg, 2004). A portion of the enrichment cultures were then streaked on MSA and BPA and incubated at 37°C for 24 to 48 h. The suspected colonies were maintained on Staphylococcal 110 (SM1110) media and kept at 4°C for identification.

Isolation and biochemical characterization of CPS

S. aureus strain (ATCC 25923) was used as a positive control for all biochemical and molecular tests. Identification of CPS was done by using selective and differential agar media. Isolates were examined by Gram stain (Bremer et al., 2004), catalase (Benson, 2002), coagulase and clumping-factor tests were carried out for the detection of staphylocoagulase (bioMe´rieux) (Winn et al., 2006) using rabbit and human plasma, mannitol fermentation (Kateete et al., 2010), oxidase (Faller and Schleifer, 1981), hemolysin production using human RBCs (Abou-Elela et al., 2009), amylase (Mishra and Behera, 2008), protease (Cheesbrough, 2002), lipase (Edberg et al., 1996), sensitivity to acriflavin (Roberson et al., 1992), lysostaphin susceptibility (Schleifer et al., 1981), acetoin production (Chapin and Murray, 1999), lecithinase (Cotar et al., 2008), tellurite reduction (Andrews, 1992), thermostable nuclease (Pascual-Anderson, 1992) and DNase test (Sánchez et al., 2003).

Molecular characterization of CPS

Extraction of genomic DNA

Bacterial culture was grown overnight on nutrient broth; 2 ml of the culture was transferred into a microcentrifuge tube and spun at 5,000 x g for 5 min. The pellet was resuspended in 567 μl of Tris- EDTA (TE) buffer to which 30 μl of 10% SDS and 3 µl of 20 mg/ml proteinase K were added. The mixture was inverted gently and incubated for 1 h at 37°C. After incubation, 100 µl of 5M NaCl was added and mixed thoroughly. Then, eighty microliter of 10% Cetyl trimethylammonium bromide (CTAB) - 0.7 M NaCl solution was added and the tubes were incubated for further 10 min at 65°C. Equal volume of phenol/chloroform/isoamyl alcohol (25:24:1) was added, mixed well and centrifuged at 10,000 rpm for 10 min. The upper aqueous phase was transferred to a new tube and an equal volume of chloroform/isoamyl alcohol (24:1) was added and centrifuged at 10,000 rpm for 10 min. The upper aqueous phase was then transferred to a new tube and 0.8 volume of isopropanol was added, mixed gently until the DNA was precipitated. The DNA was washed with 70% ethanol and resuspended in 50 μl TE buffer (Rallapalli et al., 2008).

Detection of tst, eta, and etb toxins genes by PCR

The primers used for detection of tst, eta, and etb genes are listed in Table 1 (Johnson et al., 1991). Each polymerase chain reaction (PCR) contained 2.5 μl of 10X PCR Buffer, 1.0 μM MgCl₂, 200 μM dNTP, 1U Taq DNA polymerase, 10 pmol of each primer, and 50 ng/ul of template DNA. The final volume was adjusted to 25 μl by adding sterile ultrapure water. DNA amplification was performed using the following amplification conditions: initial denaturation for 5 min at 94°C followed by 30 cycles of denaturation (94°C for 2 min), annealing (50°C for 1 min), and extension (72°C for 1 min). A final extension step (72°C for 5 min) was employed after the completion of the cycles (Rall et al., 2008).

Identification of CPS species by PCR-RFLP

Two oligonucleotide primers were used in this part: CPSK1F (CARAAYAACTGGGATTTCTGGAC) and CPSK6R (GCATCRCCRTAWGAGAATAAACG) from the highly conserved region of S. aureus kat gene sequences found in GenBank. Targeting positions were 487-509bp and 1031-1009 bp of the kat gene of S. aureus subsp. aureus Mu50 (BA000017) which allowed the amplification of 544 bp fragment. PCR amplifications were performed with a total volume of 50 μl, including: 5 μl of template DNA, 5 μl of 10X buffer PCR buffer, 2.5 μl of 50 mM MgCl₂, 0.5 μl of dNTP mix (25 mM of each dNTPs), 0.2 μl of each primer (0.1 mM), and 0.2 μl of Taq DNA polymerase solution (5 U/μl). PCR amplification conditions consisted of an initial denaturing step (95°C for 3 min) and 40 amplification cycles: a denaturing step for 10s at 95°C and an annealing-extension step for 45 sat 56°C. After amplification, 15 μl of each PCR mixtures were tested by electrophoresis on 1.5% (w/v) agarose gel at 100 V for 1 h. The remaining part (35 μl) of the PCR product was digested in a total volume of 50 μl by 20 U of TaqI restriction endonuclease at 65°C for 2 h. Restriction fragments were resolved by electrophoresis on 2% (w/v) agarose gel at 100V for 2 h (Blaiotta et al., 2010).

Identification of CPS species and subspecies by DNA sequencing

From the highly conserved region of kat gene sequences of CPS found in genbank two oligonucleotide primers were selected: CPSK1F7 (CARAAYAACTGGGATTTCTGGAC) and CPSK6R (GCATCRCCRTAWGAGAATAAACG) (Macrogen Inc. Seoul, South Korea). According to the variation in biochemical results and PCR-RFLP band patterns, strains were choice to sequence. Analysis of DNA similarity was performed using BLAST programs (Basic Local Alignment Search Tool).

Phylogenetic analysis

Using the keyword “catalase Staphylococcus”, sequences of catalase genes from different Staphylococcus species and isolates were retrieved from the National Center for Biotechnology Information (NCBI) site (www.ncbi.nlm.nih.gov). A phylogenetic tree of catalase genes was constructed using Molecular Evolutionary Genetics Analysis (MEGA) version 5.2 (Tamura et al., 2011). The Neighbor-joining (NJ) method of tree generation was used to assess the evolutionary relationships (Saitou and Nei, 1987), and the significance of clustering was evaluated by bootstrapping with 1000 replications.

S. aureus is one of the most commonly found pathogenic bacteria responsible for a broad range of nosocomial and community acquired infections due to an impressive array of toxins and other virulence determinants (Plata et al., 2009). It colonizes the skin and mucosa of human and several animal species. Although multiple body sites can be colonized in human beings, the anterior nares of the nose are the most frequent carriage site for S. aureus (Wertheim et al., 2005a). Extra-nasal sites that typically harbor the organism include the skin, perineum, and nails (Wertheim et al., 2005b). Accurate and rapid detection is important not only for choosing appropriate antibiotic therapy for the individual patient, but also for control of the endemicity of S. aureus infection.

The pathogenicity of Staphylococcus is related to the production of many virulence factors from which coagulase was considered as the most important one. In the present study, 26% of CPS was isolated from human nasal and 16% from nail samples. These results are in agreement with results obtained by other researchers (Al-Zahrani, 2012; Walther et al., 2012). However, Cross- sectional surveys of healthy adults populations have reported S. aureus nasal carriage rate of approximately 27% since 2000 (Wertheim et al., 2005a; Bischoff et al., 2004). This rate is much lower than the earlier reported prevalence of 35% which included studies since 1934 (Kluytmans et al., 1997). Improved personal hygiene and changes in socioeconomic class might explain this decline.

Strains present in the nose often contaminate the back of hands, fingers and face and so, nasal carriers can easily become skin carriers (Al-Zahrani, 2012). However, the prevalence of CPS among animal samples differed according to the sites of isolation. Nasal prevalence showed 5.4%, while meats give 16.25% (24.2 and 5% for pieces and swabs, respectively). These results were similar to the data from others (Abd El-Hamid and Bendary, 2013; Goja et al., 2013). Phenotypic characterization of CPS to species level was achieved applying growth on media supplemented with acriflavine, oxidase, mannitol utilization, hemolysin production, acetoin production, and amylase activity. However, 163 (97.6%) of CPS were characterized as typical S. aureus, while 4 (2.39%) of CPS were biochemically atypical by their production of α- hemolysin, the absence of clumping factor, and lecithinase production. The differences could be due to the diversity in the origin of the isolates (mainly animals), or might be due to some mutations that occur in the genes  thus  affecting  the  metabolic  activity   of   the species. In addition to the fact that most phenotypic identification systems have been developed for human health care and validated using clinical isolates obtained from human infections and thus might misclassify isolates from livestock (Zadoks and Watts, 2008).

Staphylococcus aureus can cause localized and invasive infections in humans. This is attributed to its ability to produce a variety of enzymes and toxins. Whereas nearly all strains of S. aureus produce enzymes that contribute to their pathogenicity, it has been generally accepted that only some strains produce ETs and PTSAgs (Bohach and Foster, 2000). In this study, the toxins genotypes of CPS were demonstrated. From one hundred and fifteen CPS isolates obtained from human source, 38.4% nasal and 26% clinical isolates possessed the gene for tst. These results are in accordance with previous findings that many healthy individuals are carriers of tst-producing strains of S. aureus (Mehrotra et al., 2000). The isolation of S. aureus strains possessing one of the pyrogenic toxins genes was previously described (Bawadi et al., 2009). In addition, half of the clinical isolates (49.3%) harbored the eta gene in comparison to (23%) for nasal. The notable higher prevalence of tst gene among clinical isolates indicates that the possession of this gene in particular seems to be a habitual feature of S. aureus. The resulted percentages are agreeable with the earlier reports and could be correlated with the transfer of this gene at high frequency (Moore and Lindsay, 2001).

On the other hand, only two human isolates harbored etb toxins genes in the nose (7.6%) in comparison to (0%) for other isolates. However, Becker et al. (2003) found that none of the clinical isolates were etb positive, while 1% of the nasal isolates were etb positive. Others also reported the absence of the genes encoding etb in the clinical isolates (Abd El-Hamid and Bendary, 2013). A geographic variation in the prevalence of different ETs isoform was reported. The majority of these reports confirmed that eta was the predominant ETs isoform in Europe, North America, and Africa which was similar to findings of this study, whereas etb-producing isolates were shown to be more frequent in Japan (Nishifuji et al., 2008). Screening for etb gene in larger samples is necessary to give better results concerning their prevalence’s in different population. The possession of more than one toxin gene was found in 8.98% of human (clinical and nasal) isolates. However, eta plus tst and eta plus etb was found in 12.17 and 0.86%, respectively. Similar coexisting pyrogenic genes combination was reported by others (Becker et al., 2003). The contribution of pyrogenic genes combination to the overall pathogenicity potential of CPS should be investigated further.

The current study revealed that the gene coding for eta toxin, was the most frequent among human than animal isolates followed by tst gene. The higher prevalence of the eta gene in staphylococci could be explained by its greater immunogenicity (Yamasaki et al., 2005). However, the absence of etb toxin gene among animal isolates of CPS indicates that the gene cannot be held responsible for the diseases that may be induced by in animal and human. Strains that expressed eta and tst genes might form an alert for public health if they pass from poultry to human. A recent study by Nemati et al. (2013) reported the absence of ETs and tst genes in S. aureus isolated from animals. However, others reported the rare prevalence of exfoliative toxins among S. aureus isolates from animals (Endo et al., 2003). This indicates that these genes cannot be held responsible for the zoonotic diseases that may be induced in human. Moreover, Adesiyun et al. (1991) reported that ETs genes were observed in 3.9% of the examined animal’s origin isolates. The present study confirms the relatively low prevalence of eta, tst encoded by genes in CPS isolated from animals and reported by others (Nemati et al., 2013). Although the importance of tst on animal health was not explained completely, it may play a role in the pathological mechanisms of bovine mastitis with its superantigenic functions (Zschock et al., 2000). Therefore, large-scale studies are required to determine the presence and role of tst in S. aureus isolates originating from livestock.

The accuracy of conventional methods for species identification and taxonomic classification of staphylococci based on phenotypic characteristics is limited (reported to be range from 50 to 70%) (Kloos and Bannerman, 1995). The use of nucleic acid targets, with their high sensitivity and specificity, provides an alternative technique for the accurate identification and classification of Staphylococcus species. Earlier results have been obtained by comparing sequences of certain genes such as hsp60, sodA, rpoB, tuf, and gap (Ghebremedhin et al., 2008). Blaiotta et al. (2010) evaluated the catalase (kat) gene performance as a new target for phylogenetic analysis of staphylococci and identification at the species level. The kat genes display a high level of restriction endonuclease polymorphism, offering good opportunities for rapid, and accurate species-level identification of staphylococcal isolates. All CPS isolates that have been identified phenotypically were confirmed genotypically by amplification of kat genes. In this study, the catalase gene of all CPS isolates was amplified by universal primers, allowing the amplification of a 544-bp region of kat containing polymorphic TaqI restriction sites of the various CPS isolates. Based on their PCR-RFLP patterns, 163 isolates were identified as S. aureus subspecies aureus, and only four isolates were identified as S. pseudintermedius. The cat gene sequence that determined in this study was similar to that already described by others (Blaiotta et al., 2010). To confirm the identification, some strains reclassified on the basis of their TaqI PCR-RFLP patterns were subjected to sequencing of the kat gene (544-bp fragment). The comparison  of  resulting  sequences  with those from reference strains indicated an agreement with those of the PCR-RFLP analysis (S. aureus subspecies aureus showed 99% homology, while S. pseudintermedius showed 82% homology to the references). It seems that the S. pseudintermedius identified in this study does not clustered with the other members of the intermedius group. S. pseudintermedius colonization is uncommon in humans, even among people with frequent contact with animals (Talan et al., 1989). They are also rare among CPS isolates from hospitalized humans (Mahoudeau et al., 1997). Their importance as a zoonotic pathogen is therefore much smaller than that of other species. However, several cases of zoonotic transmission between companion animals and humans have been reported. In some cases humans were only colonized or contaminated, but in other cases transmission resulted in human infections (Guardabassi et al., 2004).

Despites the fact that the main host of S. pseudintermedius is the dogs and cats (Moodley et al., 2014), the results of this pilot study revealed that the four strains of S. pseudintermedius were isolated from livestock including 3(5.76%) from sheep and 1(1.92%) from goats. To our knowledge, this is the first report of S. pseudintermedius strains originating from sheep and goats samples worldwide. Although, Vasil (2007) reported the isolation of S. intermedius from sheep milk samples there is a high possibility, that these isolates were S. pseudintermedius and not S. intermedius since he depends on the biochemical tests only for identification. Direct contact between animals-animals and animals-humans is a relevant factor to take into account in understanding the epidemiology and evolution of this species. Van Hoovels et al. (2006) reported the first case of S. pseudintermedius infection in a human. However, Sasaki et al. (2007) also identified two strains from humans as being S. pseudintermedius strains.

Identification of Staphylococci to species level in microbiology is important to inform therapeutic intervention and management (Geraghty et al., 2013). In human, S. pseudintermedius is an opportunistic pathogen and a leading cause of skin and ear infections, post-operative wound infections in animals mainly the dogs and cats (Weese and van Duijkeren, 2010). This species has been recognized on a few occasions as a pathogen of rhinosinusitis, a catheter-related bacteremia, and implantable cardioverter-defibrillator infections (Stegmann et al., 2010; Chuang, 2010). However, veterinary dermatologists and small animal clinical staff are sometimes considered as nasal carriers of S. pseudintermedius (Morris et al., 2010). The spectrum of S. pseudintermedius diseases have been expands which emphasizes the risk of zoonoses mainly in imunocompromised subjects (Savini et al., 2013).

Although, a limited knowledge concerning their pathogenecity was published, various virulence factors are known to be produced by this bacterium, (Fitzgerald, 2009) with the ETs as a major virulence factor (Iyori et al., 2010). This was demonstrated by the presence of 25% of the S. pseudintermedius strains that harbored eta toxin gene. By highlighting the virulence properties of the investigated strains, it has been found that all expressed high levels of protease, mannitol utilization, capable of anaerobic fermentation, and lysostaphin sensitivity. However, it is differed from S. aureus subsp. aureus by the absence of clumping-factor, and by their partial hemolytic activity (produced α- hemolysin by two strains). Unexpectedly, and although initially describing S. pseudintermedius as β -haemolytic, Awji et al. (2012) instead stated that the organism can be presumptively differentiated from S. aureus as the former lacks beta-haemolysis (using sheep blood agar). However, accurate phenotype observation remains crucial to reaching a conclusive bacterial diagnosis (Savini, 2013). Accordingly, the diagnostic algorithm of CPS should be reconsidered. It is likely that human and veterinary S. pseudintermedius isolates have been misidentified as S. aureus, S. intermedius, or other species (Bond and Loeffler, 2012). This fact should be considered when a patients' history includes contact with animals, the potential role of S. pseudintermedius as the agent of zoonoses has to be taken into account, and a correct identification may be performed. The isolation of livestock associated S. pseudintermedius in this pilot study could under line their possibility as a risk factor participating in human infections and emphasizing the need for correct species identification in clinical laboratories that handle samples of both human and animal origin. However, more useful genome-based investigations such as matrix-assisted laser desorption ionization-time of flight mass spectrometry could be used for profiling of staphylococcal strains using a large collection of staphylococci of diverse origins (David et al., 2010).

Although Livestock-associated and human-associated strains shared some virulence factors, but distinct virulence factors appeared to be important in host adaptation. Exchange of genes encoding these virulence factors between strains may expand the host range and thereby threaten public health (Fluit, 2012). More studies should be done to characterized animal isolates of CPS and prevent transferring species to health care settings. In conclusion, S. aureus subspecies aureus isolated from human seems to be different phenotypically and genotypically from livestock isolates. 

The authors have not declared any conflict of interests.