New Spa Types among MRSA and MSSA Isolates in North of Iran

Staphylococcus aureus typing using gene encoding protein A (spa typing) seems to have a high potential discriminatory power for typing this bacterium. This study was designed based on spa typing method to compare the S. aureus types among healthy carrier vs patients, and MRSA vs MSSA isolates. Method: This study was carried out on 182 spa typeable S. aureus isolates, including 52 MRSA. DNA was extracted by phenol chloroform-isoamyl alcohol method and it was amplified by specific primer of polymorphic X region of spa. Spa types were determined by Ridom Staph Type software. The spa types distribution among MRSA vs MSSA and healthy carrier vs patients, isolates were statistically compared by X2 method and P < 0.05 was considered as significant. Result: The most common types of spa in our region were t037 (18.3%) and t937 (13.9%) from 50 spa types which were identified in this study. spa types in this study were isolated from various age groups but t660 spa types were only isolated from children. Distribution of all spa types among MRSA and MSSA isolates was 16 and 38 types, respectively which is significant. In this study we found seven new spa types (belong to twelve isolates) which are reported for the first time. In 5 out of the above 7 new types the 24 bp repeated sequences in spa gene X region were already recognized but their 24 bp arrangement is introduced in our present investigation. In the remaining 2 new types we found new 24 bp nucleotide sequences which are also introduced for the first time in our present study. Conclusion: The distribution of spa types in MSSA strains was significantly higher than MRSA isolates, but there are not any specific spa types for discrimination between MRSA and MSSA. The novelty of our study is the introducing 7 new spa types including 2 new 24 bp repeated sequences in the X region of spa gene.


Introduction
Staphylococcus aureus is a well known agent as commensal organism lived on the human skin and as a leading cause of human disease.This bacterium responsible for a variety of disorders ranging from superficial skin infections up to serious abnormalities such as pneumonia, Bacteremia and Endocarditis and Staphylococcus aureus colonizes the anterior nares of 20% -80% of the human population [1] [2].
There are many molecular methods that have been employed to type, differentiate and group the S. aureus isolates, such as pulsed-field gel electrophoresis (PFGE), which have been used as the gold-standard method [3], Multilocus Sequence Typing (MLST), coa typing, phage typing and SCCmec typing.Sequence-based methods have been developed to provide fast, unambiguous, and exportable typing data.Among them, the sequence determination of the polymorphic X region of spa gene or short sequence repeat (SSR) region of the protein A gene (spa) has been proposed as an alternative to current techniques for the typing of S. aureus [4] [5].
The X region of spa gene as an epidemiological marker initially was employed in 1994 by Frenay and his colleague.The X region was amplified and its size estimated by electrophoresis [6].
In 1996, the same group improved the technique by performing sequence analysis of the X region [7].Since then many researchers have evaluated the usefulness of this technique for diverse epidemiological purposes and its credibility for S, typing.They found also it is a cost effective and simple method to apply.
Koreen et al. stated that the spa typing was shown to have a higher potential discriminatory power than other typing methods such as, microarray, multilocus enzyme electrophoresis (MLEE), Pulsed field gel electrophoresis (PFGE) and coa typing [8].Based on these later findings we chose the spa typing method to type S. aureus in our region.
The aim of this study was to compare the S. aureus spa types isolated from healthy carrier and patients, also among MRSA and MSSA isolates.

Bacterial Isolates
This study was carried out on 190 staphylococcus aureus which was isolated from 81 healthy carrier and 109 patients in Gorgan located in northern Iran during one year period .Out of this sample population, 54 (28.4%) were MRSA [9] [10].The nature of S. aureus isolates samples were confirmed using biochemical tests including catalase, cogulase, growth on MSA and DNase.

DNA Extraction
Genomic DNA for subsequent PCR was isolated from 1 ml of overnight culture, lysed with lysozym-phenol chloroform method and treated with N-lauroyl sarcosine sodium salt 2% (300 μL), proteinase k 100 μg (30 μl), and RNase A (5 μl).DNA was extracted by phenol chloroform-isoamil alcohol, chloroform, and cold ethanol.Extracted DNA were examined by electrophoris on gel agarose 1% and stored in −20˚C until experimental procedures were applied.

PCR and Spa Typing
Standard primers which was introduced by Ridom spa typing (http://www.ridom.com/spa-server/)was used for the amplification of the polymorphic X region of the protein A gene (spa).The sequences of used primer are as follow: Spa-1113f (5'-TAA AGA CGA TCC TTC GGT GAG C -3') and spa-1514r (5'-CAG CAG TAG TGC CGT TTG CTT -3') and its amplicon size varied between 300 -500 bp.
The PCR master mix and program was as follow: 1) Add genomic staphylococcal DNA in a PCR mixture to achieve 50 m L of final volume containing 1.25 units of Taq polymerase, 1.5 m M MgCl 2, 200 m M dNTPs, 0.2 m M of each primer (spa-1113f, spa-1514r) and 5 m L of 10× PCR buffer.
2) Cycling conditions consist of an initial denaturation step of 5 min at 80˚C, followed by 35 cycles of 45 s of denaturation at 94˚C, 45 s of annealing at 60˚C, 90 s of extension at 72˚C, and a final extension step of 10 min at 72˚C.The PCR product was assessed by electrophoresis on 1.5% agarose gel.

Sequencing Xr Spa
All PCR products were sequenced by company "Macrogene" in Korea.Spa types were assigned by using S. aureus Type software (version 1.4; Ridom GmbH, Würzburg, Germany), as described by Harmsen et al.

Type Ability
Among the 190 S. aureus isolates, all but 8 (4.2%) were typable by spa typing.

Diversity of Spa Types
The 182 typable isolates of S. aureus belong to 43 known different spa types and 7 new spa type which is reported for the first time in this present article.The distribution spa types among healthy carriers and patients were 32, 29 types respectively, this difference was not statistically significant (P > 0.05).On the other hand 38 types were diagnosed in MSSA isolates but only 16 spa types were present among MRSA isolates which statistically was significant (P < 0.05) (Table 1).

Distribution of Spa Type's t037 and t937
Spa type's t037, t937 with 18.3% and 13.9% were the most predominant types (Table 2).The distribution of t037, t937 between carriers vs patients and MRSA vs MSSA were not significantly different.The mean age of all subjects in this study was 31.4 ± 19.1 years, but the average age in cases particularly harboring types t37 and t937 were 39.2 ± 21.3 and 34.1 ± 15.5 years respectively, which is higher than mean age of sample population.On the other hand the spa type t660 was specifically isolated from children with mean age of 1.75 ± 1.5 years (Table 1).
Type t012 and t267 was predominantly isolated from clinical samples and all type t660 (4 cases) were isolated from patients.

Detection of New Spa Types
We found 12 isolates which have new spa types; and classified in 7 spa types.The new type 1 was the most prevalent (4 isolates) and all of them were MSSA which were isolated from patients.Apart from 2 cases which belong to new types of 6 and 7, all others cases were isolated from patients (Table 2).
In the new types 1, 2, 4, 5 and 6 the 24 repeated sequence in X region spa gene, already was recognized but the novelty this 5 types is due to how these 24 bp are arranged in gene.
But in the new type 3 and 7 we found new 24 nucleotide sequences which are introduced for the first time (Figure 1).

Discussion
Rapid and accurate determination of the different Staphylococcus aureus isolated from patients and carriers are a great help in understanding the epidemiology of this bacteria and its infection control [7].
PFGE is still considering the gold standard in molecular typing, owing to its excellent discriminatory ability [11], but the main disadvantages of this technique are related to the technical demands, the costs of the equipment, the time and labor required.Accordingly, there is a need for a rapid, inexpensive, and reliable method in routine epidemiological surveillance.Several PCR-based methods have been applied for molecular typing, for example PCR-RFLP, AP-PCR, MLST, SCCmec, coa typing and spa typing based on sequencing.In this study we used spa typing, due to its high degree of polymorphism in X region, for discrimination of different S. aureus isolated from patient, healthy carriers and between MRSA and MSSA.
In a study Nuno A. Faria and et al. (2008) used different method for typing 116 MRSA isolates and found that there were 32, 34, and 51 types in PFGE, MLST and spa methods respectively.This result indicated that discriminatory effect of Xr spa typing is more powerful even than PFGE and MLST [12].In new spa types (except 3, 7) the r types was previously determined but there arrangement is new but in new types 3, 7 we find 5 new specific 24 nucleotide sequences which marked as new.
In our pervious study PCR-RFLP method on spa gene indicated that only 8 types out of 190 isolates are S. aureus [13].In another survey conducted in India by this method only 5 spa types were detected [14], but in this present study spa typing was carried out based on its Xr region sequencing and found about 50 spa types.This means PCR-RFLP spa gene had lower discriminative effect than Xr spa sequencing method, therefore Xr spa sequence typing preferred to PCR-RFLP method of spa gene.
We found that the spa types distribution in MSSA (44 types) was significantly more than MRSA (21 types) isolates.Nuno A. Faria et al. showed spa types among 116 MRSA isolates and 82 MSSA isolates was 51 and 55, respectively (Faria et al. 2008) and Strommenger et al. (2006) in a study on 283 MSSA and 1176 MRSA isolates found 128 and 121 types, respectively [3].These latter findings are in accordance with our data which mean that the distribution of spa types in MSSA is much higher than MRSA types.
We were not able to find any spa types which we confidently label it as MRSA, although t037 was the most common type among MRSA isolates in our region but it was also found among 15 MSSA isolates.In contrast, most isolates belonging to type t937 were MSSA and a few of them belong to MRSA, and this difference was statistically meaningful.The common spa types in MRSA isolates in different region was not similar, in study conducted by Strommenger in Germany, common types were t032 and t003 in MRSA and t008 in MSSA isolates [15] and in Larry Koreen study the common type was t033 [8].Based on these latter findings the prominent spa type of S. aureus are varied in different parts of the world.
We detected that some spa types, t021, t330, t267, t436, t1149, t2313, are conclusively found among MSSA isolates.Based on our findings the question is whether this observation is due to any relation between these types and sensitivity to methicillin or it is an accidental phenomenon?Answering to this question requires a larger sample population.
The distribution of spa types among S. aureus which were isolated from healthy carrier, and patients, was similar.The most common types among healthy carriers and patients were t937 and t037 respectively.Although none of spa types was restricted to either of patients or healthy carriers, but some spa types t012, t267 and t660 predominantly detected in patients and some other spa types including t701, t779 and t2313 were more common in healthy carriers.Spa type t660 only was detected in children less than 3 years old, can this type be considered as a marker of infection on children?To answer these question extensive studies on this spa type are suggested.
We found seven new spa types in our region where out of them five types exclusively were isolated from patients, and further studies are required to address the key role played by these 5 spa types in pathogenicity, virulence and toxicity of S. aureus.

Figure 1 .
Figure 1.The sequence of 7 new spa types of S. aureus were isolated from north of Iran.*The letters A, B, G, K, M, O, Q,X and W in known spa types means a specific 24 nucleotide sequences of X region spa gene which equal to one of numbered r(r01, r08,..).In new spa types (except 3, 7) the r types was previously determined but there arrangement is new but in new types 3, 7 we find 5 new specific 24 nucleotide sequences which marked as new.

Table 1 .
Distribution of different spa types Staphylococcus aureus in North of Iran.

Table 2 .
Distribution of new spa types Staphylococcus aureus in North of Iran.