Spa Diversity among MRSA and MSSA Strains of Staphylococcus aureus in North of Iran

Protein A of Staphylococcus aureus is a pathogenic factor whose encoding gene, spa, shows a variation in length in different strains. In this study the spa gene variation in S. aureus isolated from healthy carriers and patients was studied, We also compared this variation among MRSA with MSSA strains. 208 strains of Staphylococcus aureus which we were isolated from Gorgan, north of Iran were studied, 121 cases from patients and 87 cases from healthy carriers, 59 out of them were MRSA and 149 MSSA. Samples DNA were extracted and amplified by specific primer of spa gene. In 4 (3.8%) strains of them no spa gene was detected, and 10.6% had a dual band (1200 and 1400 bp). In strains with one band, the length of spa gene differed from 1150 to 1500 bp. The most prevalent length was 1350–1400 bp (37%). The frequencies of short spa bands (1150–1200 bp) in patients strains were significantly higher. In 4 (3.8%) strains of them no spa gene was detected, and 10.6% had a dual band (1200 and 1400 bp). In strains with one band, the length of spa gene differed from 1150 to 1500 bp. The most prevalent length was 1350–1400 bp (37%). The frequencies of short spa bands (1150–1200 bp) in patients strains were significantly higher. The spa gene length of 1350–1400 bp in MSSA was more than in MRSA strains (P < .05). The average length of spa in isolated strains from urinary tract infections was more than others. It is concluded that the length of spa gene depends either on resistance to Methicillin or the source of S. aureus isolation.


Introduction
Staphylococcus aureus is one of the most important infectious pathogens in either hospitals or within the community. Protein A is a virulence factor with molecular weight of 42 KD [1]. It is covalently anchored to the peptidoglycan of S. aureus. 90% of protein A is found in the cell wall and the remaining 10% is free in the cytoplasm of bacteria. In some strains of S. aureus, protein A is unable to adhere to the cell wall and therefore is released into the media (secretary protein). This is mainly seen among meticillin-resistant S. aureus (MRSA) strains [2].
Protein A is an antiphagocytic protein that is based on its ability to bind the Fc portion of immunoglobulin G (IgG). Its NH2-terminal part contains five homologous IgG-binding units, A, B, C, D, and E, consisting of approximately 58 amino acids each. The COOH-terminal part of this protein which is thought to bind to the cell wall of Staphylococcus aureus consists of several repeats of an octapeptide. This protein acts as antiplatelet, anticomplement, and mytogen, [1,3]. It is also presented as antigen and can be detected by specific antibody in rapid diagnostic test.
Protein A has been coded by Spa gene. In Spa gene, the repeated part is located at 3 end and identified as X region; the repetitive part of region X consists of up to 12 units each with a length of 24 nucleotides. This 24 nucleotides region is highly polymorphic with respect to the number and sequence of repeats. Diversity of X region causes variation in different protein A Staphylococcus aureus [4,5]. Strain typing of Staphylococcus aureus is a good tool for epidemiologic 2 International Journal of Microbiology purpose, and many genotypic and phenotypic techniques are used to apply that. Protein A Gene, due to X repeatable area, is considered as a good one. In this research, the diversity of Spa gene in staphylococcus aureus isolated from patients and healthy carriers in this region was established and their diversity among MRSA and MSSA isolates were compared. The primary diagnosis of S. aureus based on bacterial growth on Manitol Salt Agar media, Gram Stainning, Catalase, slide or tube Coagulase and Dnase test. We used the specific primers for glutamate synthetase gene (Table 1) to confirm the bacterial diagnosis [6].

Material and Method
S. aureus resistance to methicillin was determined on the base of presence of mecA gene, using specific primers (Table 1); the amplicon size was 533 bp [7,8]. According to this method we found that from 208 isolated S. aureus, 59 (28.4%) and 149 (71.6%) were MRSA and MSSA, respectively (Unpublished data from our laboratory).
For Spa gene PCR, primer showed in Table 1 was used to identify the whole protein A genome [11].
The PCR mixture consisted of 1 mmol/L magnesiumchloride, 0.2 mmol/L dNTPs, PCR buffer, 1 µmol/L of primers, and 1 unit of Taq-DNA polymerase in a final volume of 50 µL. Samples were denaturated at 94 • C for 4 minutes followed by 35 cycles using the following parameters: denaturation at 94 • C for 1 minute, annealing at 56 • C for 1 minute, and extension at 72 • C for 3 minutes, with a final extension at 72 • C for 5 minutes [4]. In this study COL strain was used as a positive (precinct 1200 bp in Spa band), and distill water as negative control. Data were entered in spss software version 16 and analyzed with X 2 and Anova tests, and P value of <.05 was considered as significant.

Result
We did not find the Spa gene band in eight (3.8%) tested S. aureus. In 22 (10.6%) cases double bands of Spa gene were observed with the length of 1200 and 1400 bp ( Figure 1) and the reminding 179 cases (86.1%) had one band. The frequency of S. aureus isolates with no band, one band, and two bands of Spa gene, in healthy carriers, was 5.8%, 88.4%, and 5.8%, and in patients, 2.5%, 83.5%, and 14%, respectively (P = .088). But the frequency of strains having two bands in patients are significantly more than carriers (P = .042). Three strains of S. aureus without Spa gene were isolated from patients, and the urine was the source of these bacteria and this phenomenon were not observed in any other clinical sample.
Majority of MRSA strains that have one band of protein A gene, have the length band between 1150-1200 bp, while most frequencies in MSSA strains have the length band between 1350-1400 bp. The differences in length is significant between two groups of MRSA and MSSA (P < .001) ( Table 3).  The Prevalence of strains with two bands of Spa gene in isolated from wounds (21.4%) and urine (16.7%) was more than in other clinical samples, and the most cases with a short band of Spa gene (1150-1200 bp) were separated from wounds and blood infection. According to Table 4, the average length of Spa gene in strains which were isolated from urinary tract infections is more than other strains.
The average ages of people who were infected with S. aureus isolates which consisted of two bands, one band, and no band were 17.4 ± 30.2, 19.5 ± 32.8, and 8.4 ± 35.5 years, respectively, but this difference is not meaningful statistically (P > .05). There is no statistical difference between the Spa gene types among S. aureus isolated from males and females (P > .05).

Discussion
In this study, 8 isolates of Staphylococcus aureus (3.8%) had no Spa gene band, and most of them were observed in strains which were isolated from healthy carriers; therefore frequency of protein A gene was about 96.2% (96.6% in MRSA versus 96% in MSSA). In other studies, the prevalence of S. aureus without protein A expression is reported to be up to 5% [12], but different studies on Spa gene showed a lower frequency of strains without this gene. Faria and colleagues showed that 99% of Staphylococcus aureus strains can be typed with Spa gene [13]. Also Strommenger showed that in 1459 strains of Staphylococcus aureus, 99.8% strains are typeable by Spa gene typing [11] these later findings seem to be lower than our results. We could not find any documented report in the literature working with PCR method on Spa gene and demonstrating gene with double bands. Our study is the first documented report of the strains of S. aureus with two Spa gene. The frequency of this phenomenon is 10.6% with the length of 1200 and 1400 bp.
In addition to standard diagnostic methods we tested all S. aureus isolates by specific glutamate synthetase (sa442); we also carried out the Spa gene PCR method in triple times and identical results were found; due to this observation we argue strongly in the favor of new strain of S. aureus in our regions. This strain is more prevalent in patients than healthy carriers and it is more common in MRSA than MSSA. The more accurate identification of this strain and its importance is necessary.
Our study has showed that the length of Spa gene in MRSA strains was significantly shorter than MSSA strains. This issue indicated that the number of repetitive sequences in Xr Spa in MSSA strains is more than in MRSA strains. Frequencies of strains with short Spa bands (1150-1200 bp) in strains isolated from patients were significantly more than from healthy carriers. This can be due to the requirement of bacterial attachment to the nasal epithelial cells in healthy carriers by a longer protein A. We argue that S. aureus strains with shorter length of protein A cannot adhere to the surface of nasal epithelium and is discharged by breath, sneezing, and coughing [14].
Protein A has a recognized pathogenic role in S. aureus, and this process is via the anchoring to Fc Domain IgG, Complement fixation, and so forth. This protein also has a role in attachment and invasion of target cells, joung and colleagues (2001) show the role of this protein in KB cells connection [15].  Although the length of Spa gene do not have a meaning correlation with the type of infection; our result showed that the length of Spa gene in strains which are isolated from urinary tract infection is more than samples from blood, wounds, and other clinical samples. This observation may be due to the role this protein plays in conjunction or stronger connection of bacteria to the urinary tract epithelial cells [16].

Conclusion
Our study showed that 3.8% of Staphylococcus aureus strains in Gorgan (north of Iran) have no Spa genes. About 10% of them were consisted of dual-band Spa in PCR. The majority of our strains showed to have the length of Spa gene between 1350-1400 bp. Frequencies of strains with short Spa bands (1150-1200 bp) in strains isolated from patients were significantly more than those isolated from healthy carriers. The average length of Spa gene in strains which were isolated from urinary tract infections was more than other clinical strains. It is concluded that the length of Spa gene depends either on resistance to methicillin or the source of S. aureus isolation.