Determining spa-type of methicillin-resistant Staphylococcus aureus (MRSA) via high-resolution melting (HRM) analysis, Shiraz, Iran

Objectives Molecular typing methods are useful for rapid detection and control of a disease. Recently, the use of high-resolution melting (HRM) for spa typing of MRSA isolates were reported. This technique is rapid, inexpensive and simple for genotyping and mutation screening in DNA sequence. The aim of this study was to evaluate the ability of HRM-PCR to analysis spa genes amongst MRSA isolates. Results A total of 50 MRSA isolates were collected from two teaching hospitals in Shiraz, Iran. The isolates were confirmed as MRSA by susceptibility to cefoxitin and detection of mecA gene using PCR. We used HRM analysis and PCR-sequencing method for spa typing of MRSA isolates. In total, 15 different spa types were discriminate by HRM and sequencing method. The melting temperature of the 15 spa types, using HRM genotyping were between 82.16 and 85.66 °C. The rate of GC % content was 39.4–46.3. According to the results, spa typing of 50 clinical isolates via PCR-sequencing and HRM methods were 100% similar. Consequently, HRM method can easily identify and rapidly differentiate alleles of spa genes. This method is faster, less laborious and more suitable for high sample at lower cost and risk of contamination.


Introduction
MRSA is one of the most common nosocomial pathogens worldwide, leading to severe morbidity and mortality. In Staphylococcus aureus, the mecA gene causes resistance to methicillin. This gene produces PBP2A (protein binding to penicillin) which inhibits beta-lactam antibiotics [1]. Molecular typing methods are useful for rapid identification and to control disease [2]. Nowadays, pulsedfield gel electrophoresis (PFGE) is the gold standard method for the typing of S. aureus isolates; however, this method is time consuming process, expensive, complicated, and difficult to standardize [3]. Amongst molecular typing methods, SCCmec typing method is simple and cost effective with high discriminatory power to detect MRSA isolates associated with nosocomial infections [3]. In recent years, molecular typing of S. aureus has been performed by spa sequence typing [4][5][6]. The Staphylococcus protein A (spa) gene has the polymorphic in X region, which determines by sequencing method [5,6].
In recent years, high-resolution melting (HRM) analysis has been used to analyze genetic variations. This technique is rapid, inexpensive, simple and cost effective for genotyping and mutation screening in DNA sequence

BMC Research Notes
*Correspondence: hadina@sums.ac.ir [7]. HRM with real-time polymerase chain reaction (PCR) can be used for spa typing hypervariable X region of the spa gene [7][8][9][10][11]. In this technique, DNA strands are distinguished based on the length and percentage of the GC content [11]. The aim of this study was to assess the ability of HRM-PCR in analyzing spa genes amongst MRSA isolates.

Bacterial isolates
In this study, from January 2017 to June 2018, a total of 50 MRSA isolates were collected from different patients. All isolates were identified as MRSA by standard biochemical tests, resistance to cefoxitin and detection of mecA gene, as it was described previously [12].

High-resolution melting PCR method
The genomic DNA was extracted using Exgene Clinic SV (GeneALL, Seoul, Korea) according to manufacturer's guidelines and primer pair spa (spa-1113f and spa-1514r) were applied to the HRM method.
The HRM analysis was performed by the Quant studio real-time PCR system (ABI bio system, USA). HRM was performed in 25 µL reaction containing 5 µL of 5 HOT FIREPol EvaGreen HRM Mix (ROX) (Solis BioDyne Co, Estonia), 1 µL of each primer (10 pM), 2 µL of DNA template (20 ng) and 16 µL of water (DNase and RNase free water, SinaClon BioScience Co, Iran). The PCR thermocycling condition was: 95 °C for 12 min, 40 cycles at 95 °C for 15 s, 63 °C for 20 s and 72 °C for 20 s. Then, 95 °C for 1 min and 40 °C for 1 min followed by HRM ramping from 75 to 90 °C with fluorescence data acquisition at 0.5 °C increments. The melting curves of all samples were analyzed by HRM Software v3.0 (Thermo Fisher Scientific Co, USA).
Using conventional PCR-sequencing analysis for the 50 clinical isolates of MRSA, showed 15 different spa types, including spa types; t021, t018, t030, t037, t386, t081, t325, t345, t790, t314, t186, t304, t003, t1877, t1816. Spa type t030 was the major type in this study. These spa types were defined as the control for HRM analysis. The processing time for HRM analysis was around 2 h. The melting temperature of the 15 HRM genotypes was between 82.16 and 85.66° C. The rate of GC % lies between 39.4 and 46.3 ( Table 1). Typing of the 50 clinical isolates by the PCR-sequencing method and HRM method resulted in 100% similarity (Table 1).

Discussion
MRSA can cause infections both in hospitals as well as communities [13,14]. Due to outbreaks of MRSA increase in the mentioned settings, typing methods are vital to systematically control these infections. Molecular typing methods of microbial pathogens are important tools for detecting the outbreaks, controlling and preventing infections as well as for the epidemiological surveillance [5]. Different typing methods are done for epidemiological investigations such as PFGE, multilocus sequence typing (MLST), spa typing and SCCmec typing [14]. PFGE and MLST are appropriate typing methods; however, these methods are expensive and time-consuming [15]. Spa typing method is based on short sequence repeats of hypervariable X region in the spa gene, but this method is still expensive. But then again this typing method is effective and easy technique to detect typing MRSA isolates [16]. In a previous study, we found 15 different spa-types, and spa type t030 was the most common amongst MRSAs [17]. HRM analysis has been recently introduced as an appropriate method for epidemiological investigations and detection of sequence variants in clinical research and diagnostics. HRM method can easily and rapidly identify the different alleles of spa genes [11]. This method is used in a closed system and in the same tube as the amplification step; hence, it is faster, easier, and more suitable for high sample, which reduces the risk of contamination [18]. In our study, the results of HRM showed 15 different spa types. All of the isolates had significant difference in Tm and GC content. The G + C percentage and the length of the tandem repeats in the X region of spa gene might cause differences in melting temperature among the samples. Our spa typing results was completely similar to HRM results. This finding is in line with the findings of Hon-Kwan Chen et al. study in China [8]. However, our results were in contrast with the results of Fasihi et al. [11] and Stephens et al. [10], which showed that for some isolates, Tm HRM with conventional PCR sequencing were different. HRM is a suitable method to be used to examine hypervariable loci, since it can provide more clearance for genotyping and diversity among MRSA strains. Also, another study claimed that this technique is less expensive than spa sequencing [19]. Spa typing using with other typing methods, such as MLST and SCCmec can be distinguished better amongst MRSA clones in hospital [19]. Different values of melting temperature were reported for one spa type. HRM method can be affected by testing conditions and type of real time machine as well as HRM software [9,11]. In this study, Tm value for t030 was 83.83 °C, but in a previous study by fasihi et al. it was 85.3 °C. Also, in the present study as well as a previous study by Fasihi et al. HOT FIREPol EvaGreen HRM Mix (ROX) was used, but in another study Platinum SYBR-Green was used [9]. In our study, using HRM technique, we used Quant studio real-time PCR system (ABI bio system, USA) and 5 × HOT FIREPOL EvaGreen HRM Mix (ROX) (Solis Bio Dyne Co, Estonia) while in another study, a different real time machine and master mix was used [8,9]. These differences can be as result of using different real time PCR device or Master Mix producing different TMs for the same spa type. Even though HRM method is simple, and more economical than the conventional method, it has several limitations. The HRM method cannot be replaced by sequencing and it requires the presence of positive controls for each polymorphism.
In this technique it is essential to have a known control type; however, if there is a new spa type conventional PCR-sequencing is required. Also, isolates that have the same GC content with the same TM, their discrimination is difficult [8]. In HRM the basis of differentiation is fragment length, sequence, and GC percentage. So, as mentioned above, the greater the difference, it will be possible to separate and differentiate is greater too and vice versa.

Conclusion
HRM method is simple, more economical than the conventional method. HRM method can easily and rapidly identify the different alleles of spa genes. This method can analysis faster is less laborious, more suitable for high quantity samples with lowers risk of contamination. Accordingly, this technique has the potential to show variation, but require control, and should be accompanied by the spa gene sequence, and it is not suitable for new strains.

Limitations
A limitation in this study was that we did not carry out HRM for MSSA isolates, due to financial constraints.