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Introduction
Staphylococcus aureus is a major etiological pathogen of bovine mastitis, which triggers significant economic losses in dairy herds worldwide. S. aureus produces a variety of virulence factors that are responsible for subclinical and persistent intra-mammary infections [12]. S. aureus associated mastitis is one of the most contagious diseases presently plaguing dairy herds [25]. Investigating distributions and the virulent factors of S. aureus provides important information for establishing infection control strategies.
Among the various molecular typing methods for S. aureus, spa typing that targets the spa gene is an effective and rapid method [36]. The spa gene possesses a repetitive region called the short sequence repeat (SSR), which consists of a variable number of 21-27-bp nucleotide repeats. A spa typing is performed by analyzing the number and sequence of the repeats. Since results of spa typing agree with those of pulsed-field gel electrophoresis (PFGE), implicating spa typing as a useful alternative genotyping method [39]. Moreover, the clonal relation between isolates can be investigated by clustering related spa types using the Based Upon Repeat Patterns (BURP) algorithm [24].
Many mastitis associated S. aureus strains produce staphylococcal enterotoxin (SE) or SE-like toxin that are part of the superantigen (SAg) family [4,26]. SAgs manifest their virulence by ligation of the major histocompatibility complex class II molecules and the Vβ chain of the T-cell receptor from the outside. This leads to the stimulation of T-cell proliferation in a nonspecific manner, ultimately causing the host immune system to be suppressed [7]. There are 18 kinds of SE and SE-like toxin subtypes, which are defined based on the amino acid homology [23,29,40-43]. Each toxin subtype is assumed to be related with a host-specific immune reaction [8]. Thus, SE gene profiles, the detected SE and SE-like toxin genes of S. aureus isolates, can be used for investigating the role of those toxins in bovine mastitis.
Most of the genes encoding SE and SE-like toxin reside on the mobile genetic elements (MGE) including plasmids, prophages, staphylococcal pathogenic islands (SaPI) and genomic islands (νSa) [3,23,27,33,35]. Horizontal transfer of MGE is an important means for acquisition of virulence factors by S. aureus [3,14]. Several studies have predicted the MGE possessed by S. aureus isolates based on the SE gene profiles [16,28].
In this study, S. aureus strains isolated from the milk of cows suffering mastits in Korea were investigated by spa typing and SE gene profiling. From the spa typing, genotypes and geographical distributions of S. aureus in five provinces were determined. Moreover, SE gene profiling revealed the prevalence of each subtype of SE or SE-like toxin genes and the associated MGE related with those genes.
Materials and Methods
Raw milk sampling
From 2006 to 2007, a total of 13,955 raw milk samples were collected from 922 dairy herds in Gyeonggi, Gangwon, Chung-cheong, Jeolla, and Gyeong-sang provinces. Samples were aseptically collected from individual quarters of the cows as previously described [26]. Somatic cell counts (SCC) of milk samples, which indirectly show the quantity of dead epithelial cells and neutrophils in the milk, were analyzed using a somatic cell counter (Bentley Somacount 300; Chaska, USA). Milk samples with over 500,000 cells/mL of SCC were selected for bacterial isolation [31].
Bacterial isolation and DNA extraction
S. aureus strains were isolated from the suspected mastitic milk. In brief, 10 µL of milk was inoculated onto 5% sheep blood agar (Promed, Korea), which was incubated at 37℃ for 24 h. Colonies showing characteristic phenotype of S. aureus were sub-cultured on Baird-Parker agar (Becton Dickinson, USA). S. aureus was identified through Gram staining and catalase, oxidase, DNase, TNase and VP tests. Confirmation was provided by PCR targeting the S. aureus-specific nuc gene [5].
Genomic DNA was isolated using a DNeasy tissue kit (Qiagen GmbH, Germany) according to the manufacturer's instructions with a modification of the cell lysis step performed with 50 U/mL lysostaphin (Sigma-Aldrich, USA). The concentration of the isolated DNA was estimated using Specgene (Techne, UK) at A260, and the DNA was diluted to a concentration of 50 ng/µL.
spa typing
The spa typing was preformed as previously described [15]. Briefly, the SSR region of the spa gene was amplified using primers 1095F (5'-AGACGATCCTTCGGTGAGC-3') and 1517R (5'-GCTTTTGCAATGTCATTTACTG-3'). Sequence analyses of extracted PCR products were performed at Bionics (Korea). Sequence data were analyzed using Ridom StaphType software (Ridom GmbH, Germany) which automatically detects the spa-repeats. The spa repeats and spa types were determined and assigned a numeric code using a previously described method [15]. BURP clustering for the spa types were performed with Ridom StaphType software (Ridom GmbH, Germany).
Multiplex PCR detection of SE and SE-like toxin genes
Multiplex PCR was performed to detect 18 kinds of SE and SE-like toxin genes (sea to see, seg to sej, selk to selr, and selu) as previously described [17]. Each PCR reaction was performed with 1 µL of the prepared template DNA, 10 µL of ×5 primer mixture (0.5 µM of each primer), and 25 µL of ×2 Multiplex Master Mix (Seegene, Korea), with the final volume adjusted to 50 µL with distilled water. PCR was performed using the following steps: 94℃ for 15 min, 30 cycles of 94℃ for 30 sec, 55℃ for 30 sec, and 72℃ for 2 min with a final extension at 72℃ for 10 min. The amplified PCR products were resolved by electrophoresis in 2% agarose gel (Sigma-Aldrich, USA) at 100 V for 60 min.
Results
spa types and geographical distribution of S. aureus
A total of 44 S. aureus strains were isolated from 26 herds in five provinces of Korea (Fig. 1). The positive rate of S. aureus detection over the regions was 2.82 ± 0.11% of screened dairy herds and 0.32 ± 0.02% of collected milk samples.
Table 1 shows the spa type and the geographical distributions of all isolates. A total of 14 different spa types were identified. All had 4-11 repeats and the numeric code of the spa types were assigned based on the repeat pattern. Several spa types showed similar repeat patterns, suggesting they had diverged from a common ancestor by the accumulation of point mutations or the rearrangement of spa repeats. These were grouped together into the same cluster using the BURP algorithm. The similar spa types were assorted into one cluster; each cluster displayed an evolutionary relationship between S. aureus isolates. For example, isolates t148 and t324, which are different only the r21 repeat, were included in the same cluster and it was assumed that those two isolates were propagated from the same ancestor. In total, the isolates were grouped into five clusters and two singletons based on their spa types. There was no herd where more than one spa type was detected. Seventeen isolates (38.6%) were included in cluster 1, distributed in all five provinces, and 16 isolates (36.4%) were included in cluster 2, distributed in four of the five provinces. The other minor clusters and singletons were distributed among one or two provinces.
SE gene profiling
SE and SE-like toxin genes were detected in 34 (77.3%) isolates among the 44 isolates (Table 2). Of the 18 kinds of SE and SE-like toxin genes, 10 genes were detected; sea (n = 2 isolates), seb (n = 1), seg (n = 17), seh (n = 16), sei (n = 17), selk (n = 2), selm (n = 17), seln (n = 17), selo (n = 17) and selq (n = 2). There was no detection of sec, sed, see, sej, sell, selp, selr and selu genes.
Six SE gene profiles were analyzed based on detected SE and SE-like genes in the S. aureus isolates (Table 2). The most prevalent SE gene profile was a composition of seg, sei, selm, seln, and selo genes, which was detected in 16 isolates (36.3%). Interestingly, this profile was the same as the SE and SE-like toxin gene composition of the Type I νSaβ genomic island [3]. Likewise, the other profiles were also comparable to the SE and SE-like toxin composition of previously reported MGE such as prophage φSa3mws (sea, selk, and selq) [3], pathogenic island SaPI3 (seb, selk and selq) [42] and prophage φSa3mu (sea) [23].
The SE gene profile and spa type of S. aureus isolates were also compared (Table 2). Several spa types showed more than two SE gene profiles. Among the eight isolates of the t164 type, seven isolates possessed seg, sei, selm, seln and selo genes, while the remaining isolates harbored none of the toxin genes. The eight t286 strains had two SE profiles; seh gene positive isolates (n = 6) and toxin negative isolates (n = 2). In the case of t127, seven isolates harbored the seh gene and one isolate harbored the sea, selk, selq and seh genes. The t189 strains displayed three kinds of SE gene profiles: three isolates were suspected to possess the seg, sei, selm, seln and selo genes, two isolates possessed the seh gene, and two isolates did not harbor the toxin genes.
Discussion
Investigating distributions of S. aureus in dairy herds is important for establishing infection control strategies by providing basic information. Over the past decade, many epidemiological studies for S. aureus mastitis have been performed using various molecular typing methods. In the present study, the genotypes sand geographical distribution of S. aureus isolates were investigated in a rapid, unambiguously and cost-effective fashion using spa typing.
PFGE is the gold standard for genotyping because it is based on the whole genome of the isolate. Nonetheless, it is time-consuming to do and inter-lab comparison is onerous. Therefore, DNA sequence-based approaches have been suggested as an alternative typing method. Multilocus sequence typing (MLST), which is based on the sequences of seven housekeeping genes in S. aureus, has been successfully adapted to S. aureus [9]. However, MLST is not suitable for routine surveillance due to its high cost. In contrast, spa typing is more cost-effective because it targets only a single locus of repeat regions.
The results of spa typing can be easily compared between laboratories. A survey of Brazilian bovine S. aureus isolates revealed spa types of t359, t267 and t605 [1]; these isolates were not presently detected. On the other hand, spa types t127, t148, t324, t1151, t002 and t008 observed in this study have been reported human S. aureus isolates in Korea [32]. As a result, the distribution of S. aureus might be geographically dependent. The present prevalence of each bovine type differed from the Korean human S. aureus isolates. For example, t002, which is the predominant human type [32], was presently observed in only one isolate.
The other advantage of spa typing is the rapid clustering that can be accomplished using the BURP algorithm. The most significant epidemiological finding of this investigation was that predominant clusters were widespread over the provinces, although other minor clusters were only locally distributed. This agrees with a previous survey [26]. Several studies also revealed the distribution of only a few specialized clones among dairy herds [1,2,11,19,20]. The predominant strains may be subclones, which were a source of contagious transmission, or were host-adapted [34].
Moreover, the predominant clusters consisting of several spa types might have a lengthy history in bovine mastitis ecology [6], since considerable time is required for the accumulation of changes on spa gene repeat sequences [13, 22]. On the other hand, spa types classified as singletons may have been more recently introduced. These findings imply that country-wide control systems are needed to prevent the spreading of a contagious strain between herds. Additionally, genetic backgrounds and molecular mechanisms of these predominant S. aureus strains should be investigated
In this study, 77.3% of the S. aureus strains possessed the SE or SE-like toxin genes. This supports the suggestion that SE and SE-like toxins may cause bovine mastitis by depressing the bovine immune system [10]. The most prevalent SE or SE-like toxin genes were seg, sei, selm, seln and selo, which were detected in 38.6% of the S. aureus isolates. This result agrees with a previous study [37] that reported the high prevalence rate of recently described SE or SE-like toxin genes among S. aureus strains isolated from bovine milk. The prevalence of those newly reported SE and SE-like toxin genes and their role in a bovine mastitis should be investigated further.
Among the classical SE genes, sea to see, sea showed the highest prevalence rate in the present study. This is a cause of concern as a potential health risk for humans, because most S. aureus strains that possess the sea gene produce the SEA toxin, which is a major etiological factor of staphylococcal food poisoning [4]. On the other hand, it is interesting that no sec gene was detected, although sec gene detection from animal isolates has been described [21,30,38]. This may suggest a difference between geographical and periodical distributions of enterotoxigenic S. aureus.
The SE gene profile of seg, sei, selm, seln, and selo was always detected together in this study, and this composition exactly matched that of Type I νSaβ. This strongly suggests that S. aureus isolates possessing those genes might be associated with Type I νSaβ. The other presently-described SE gene profiles are also comparable to various MGE. Moreover, the SE gene profile differences within the same spa types support the possibility of MGE association. For example, φSa3mw might be associated with the t127 isolate possesses sea, selk and selq in addition to the seh gene, unlike the other seven t127 isolates. It is also possible, however, that the SE gene profile of sea, seh, selk, and selq might be associated with φSa3mu (sea) and that the seh, selk, and selq genes are encoded on a chromosome or other unknown type of MGE. More genetic investigations will be necessary to clearly reveal the association between SE profiles and MGE of S. aureus isolates.
This study is a first report of spa types and the prevalence of the recently described SE and SE-like toxin genes among S. aureus isolated from bovine raw milk in Korea. From the spa typing, it was shown that there were two predominant S. aureus clusters distributed widely in bovine husbandry: cluster 1 composed of t164, t2094 and t1987, and cluster 2 composed of t286 and t127. The transmission and pathogenesis of the predominant strains and the relationship between bovine mastitis and recently described SE and SE-like toxins will be investigated further.
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