Comparison of HRM analysis and three REP-PCR genomic fingerprint methods for rapid typing of MRSA at a Brazilian hospital

Introduction: Infections caused by multidrug-resistant bacteria are increasingly common and represent a serious problem for public health. Staphylococcus aureus is one of the major agents of infections, and methicillin-resistant S. aureus (MRSA) has spread worldwide. The aim of this study was to phenotypically and genotypically characterize 55 MRSAs isolated in the University Hospital of Londrina, Paraná, Brazil, during 2010. Methodology: Bacterial isolates were characterized based on their antimicrobial susceptibility profile, biofilm production capacity, and staphylococcal chromosome cassette mec (SCCmec) type. Determination of clonal groups was performed by polymerase chain reaction using the RW3A, JB1, and BOX A1R primers and high-resolution melting (HRM) analysis. Results: The majority of isolates harbored SCCmec type II. SCCmec III, characteristic of the Brazilian endemic clone, was observed in four strains. Only two isolates harbored SCCmec type IV, which is common in community-acquired MRSA strains. Most isolates also showed resistance to more than four of the tested antimicrobials, and 30 isolates exhibited the ability to produce biofilm. DNA polymorphism analysis showed a higher discriminatory power for the JB1 primer, but RW3A revealed several clonal groups of MRSA with similar genotypic and phenotypic characteristics. HRM analysis showed eight different sequence types. Conclusions: These results are important for epidemiological studies involving MRSA infections.


Introduction
Staphylococcus aureus is a major cause of infections in both healthcare facilities and the community, causing pneumonia, bacteremia, endocarditis, osteomyelitis, abscesses, and septic arthritis.
The Centers for Disease Control and Prevention (CDC) of the United States reported that more than 80,000 illnesses and 11,000 deaths in the hospital setting were caused by MRSA during 2011.Compared to earlier periods, there was a 31% decline in global rates of invasive MRSA infections [2].The largest decrease (54%) was observed in infections in patients during hospitalization; this was due to preventive measures, which are important for controlling outbreaks [2].
Both healthcare-associated MRSA (HA-MRSA) and community-acquired MRSA (CA-MRSA) exhibit resistance to beta-lactam antimicrobials.However, CA-MRSA strains are typically less resistant to other classes of antimicrobials.Oxacillin resistance is encoded by the mecA gene, which is located on the staphylococcal chromosome cassette mec (SCCmec).Overall, HA-MRSA strains are classified as SCCmec I, II, or III, whereas CA-MRSA strains containing SCCmec types are generally IV, V, and VI.CA-MRSA strains may produce Panton-Valentine leukocidin (PVL) toxin, which has been associated with skin abscesses and necrotizing pneumonia [1].
The genetic relatedness of MRSA isolates has been analyzed by different methodologies.The gold standards for this analysis are pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), and spa typing, but these techniques are expensive, time consuming, laborious, and not accessible to all research laboratories [19][20][21].
Polymerase chain reaction (PCR)-based methods for analyzing the genetic relatedness of MRSA strains offer the advantage of being faster and easier to perform [22].In general, repetitive sequences with unknown function are present in genomic DNA from different bacterial species [23], and these regions have been used to determine the DNA genetic similarity between several bacterial species, including MRSA [24][25][26].
The RW3A, JB1, and BOX primers were initially used to evaluate the clonality of some bacteria.The RW3A primer was derived from a Mycoplasma pneumoniae repetitive sequence (RepMP3) [27] and has been applied in studies with MRSA [25,26].The JB1 primer was sequenced from Enterococcus faecium and found to be useful for differentiating E. faecalis strains [28], another bacteria that can transfer resistance encoding genes to S. aureus.BOX elements contain sub-sequences that are differentially conserved, representing the first repeating element from Streptococcus pneumoniae [24], and have been used to characterize different species of Staphylococcus [29].
High-resolution melting (HRM) analysis of singlenucleotide polymorphisms (SNPs) of small fragments derived from the MLST database has been also used for genotyping MRSA.This technique was described by Lilliebridge et al. [30], performed with real-time PCR with small fragments derived from MLST and is capable of verifying the MRSA sequence type [31].
Some advantages of the techniques used in these experiments are their low cost, rapidity, and use of the instrumentation available in laboratories.
The aim of this study was to characterize MRSA isolates from patients seen at the University Hospital of Londrina, Paraná, Brazil, during 2010 using phenotypic and genotypic methods.All isolates were tested for antimicrobial susceptibility, SCCmec typing, and biofilm-forming capacity.In addition, the genetic diversity between the isolates was analyzed by PCRbased methods using SNP DNA as the target, and HRM analysis.

Bacteria
MRSA isolates were isolated from patients hospitalized at the University Hospital of Londrina between January and September 2010.The bacteria were deposited in the bacterial collection of the Department of Pathology, Clinical and Toxicological Analysis from University Hospital of Universidade Estadual de Londrina (UH/UEL).A total of 55 isolates were obtained from blood (n = 15), urine (n = 4), tracheal aspirates (n = 9), and general discharge (n = 27), such as tissue secretion, bone fragment, and peritoneal fluid.MRSA BEC 9393 [32] and N315 [33] isolates were provided by Dr. Agnes Marie Sá Figueiredo (Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil) and Dr. Elsa Masae Mamizuka (Universidade de São Paulo, São Paulo, Brazil), respectively.Methicillin-sensitive S. aureus (MSSA) ATCC 25923 and 29213 were used as reference strains.These reference strains were provided by Dr. Marcelo Brocchi (Universidade Estadual de Campinas, Campinas, SP, Brazil).All bacteria were stored at -80°C in brain-heart infusion (BHI) (Difco, Detroit, USA) plus 25% glycerol (Sigma, Poole, UK).
The protocols of this study were in accordance with the National Research Council and were approved by the ethics and human research of UEL (protocol 186/09 CEP/UEL).
For urine isolates, CLI and ERY were not evaluated because they are not used for the treatment of MRSA urinary tract infections.
These tests were in accordance with the criteria established by the CLSI [34].S. aureus ATCC 29213 and Enterococcus faecalis ATCC 51299 were used as controls.
The PCR products and a molecular DNA weight marker X (0.07-12.2 kb) (Roche, Indianapolis, USA) were separated by electrophoresis in 2% agarose gel (Invitrogen, Carlsbad, USA) at 75 V and visualized on a UV transilluminator after staining with Gel Red (Biotium, Hayward, USA).
The cluster analysis was performed using BioNumerics version 4.6 (Applied Mathematics, Kortrijk, Belgium) using the UPGMA algorithm and the Jaccard coefficient (J) [38] with a tolerance of 3%.

High-resolution melting PCR method
The isolates used for this analysis were chosen based on different genotypes obtained from RW3A and JB1 primers analysis.One to three samples from each clonal group were selected.The real-time PCR reaction and HRM analysis were done according to Lilliebridge et al. [30].The SNPs of fragments derived from arcC78/210, aroE88/155, gmk286, pta294, tpi36, and tpi241/243 were analyzed following the methodology described by Lilliebridge et al. [30].The HRM analysis was performed using the Rotor-Gene Q 5Plex HRM System (Qiagen, West Sussex, UK) using a Type-it HRM PCR Kit (Qiagen, West Sussex, UK).For each separate run, three control strains with known ST type (ST239, ST5, and ST243) were included as melting curve standards.Reactions were carried out in duplicate.
The melting curves of all samples were generated automatically by the Rotor-Gene Q software, version 2.1.0.9 (Qiagen, West Sussex, UK) and normalized using the default setting.HRM difference plot was also generated by the software, which could give a better comparison of melting temperatures among different MRSA ST types.

Biofilm test on a polystyrene surface
The MRSA isolates were tested for biofilm production on a polystyrene surface stained with crystal violet according to Stepanovic et al. [39].MRSA BEC 9393 and culture medium alone were used as the positive and negative controls, respectively.The optical density (OD) of each well was measured at 570 nm using an automated plate reader (Synergy HT, Biotek, Winooski, USA).
The mean value of the OD of the negative control (ODc) was used for comparison with those obtained from the isolates (ODi).The ODi of each isolate was obtained by the mean minus the ODc.The isolates were classified as non-biofilm producers when ODi ≤ ODc and as biofilm-producers when ODi > ODc.

Statistical analysis
Significant differences among the results obtained were examined using the Fisher method.Differences were considered significant at p < 0.05 using BioEstat version 5.0 software.

Patients, antimicrobial susceptibility profile, and species identification
The age of the patients enrolled in this study ranged from three months to 79 years (median of 48 years), and the majority of them were men (n = 43; 78.18%).
All MRSA isolates in this study showed the presence of coa and mecA genes, FOX resistance, and LNZ susceptibility.Among the other antimicrobial agents tested, resistance to CIP (94.5%),ERY (94.1%), and CLI (92.2%) was most prevalent in these isolates.However, the percentages of SXT (29.1%),RIF (23.6%), and TET (16.4%) resistance were lower than that of the other antimicrobials.
Based on the antimicrobial resistance profiles (number and type of antimicrobials), seven antibiotypes (A, B, C, D, E, F, and G) were established for these isolates.Antibiotype A was resistant to CIP, CLI, ERY, GEN, SXT, TET; antibiotype B was resistant to CIP, CLI, ERY, GEN, SXT; antibiotype C was resistant to CIP, CLI, ERY, GEN, RIF; antibiotype D was resistant to CIP, CLI, ERY, GEN; antibiotype E was resistant to CIP, CLI, ERY, RIF; antibiotype F was resistant to CIP, CLI, ERY; antibiotype G was resistant to ERY; and antibiotype O was resistant to CIP and some others.

SCCmec typing
SCCmec type II was the most prevalent, being present in 24 isolates, followed by SCCmec type I, which was found in 19 isolates.SCCmec types III and IV were found in 4 and 2 isolates obtained from general discharges, respectively (Table 1).Four isolates (MRSA 102, 109, 119, and 122) showed SCCmec type III, equivalent to MRSA BEC9393, and these isolates showed the A antibiotype.Six isolates were not typeable by the technique used here.

Clustering of genotypes of SNPs
Applying HRM analysis, eight different STs between 21 isolates were found.ST5 was the most frequent and was present in 10 isolates, displaying the same ST of S. aureus N315.STs from control were based on reference strains.Results of all isolates are shown in Table 2.
Although few isolates had been selected from each clonal group detected in the RW3A-JB1 methodology, there was no correlation with the clonal groups in the HRM methodology.

Biofilm
Among the 55 isolates, 30 produced biofilm on the polystyrene surface, and none of the seven catheter isolates showed biofilm production (Table 1).Some isolates produced more biofilm than others, with results that were four times greater than ODc, being the strongest biofilm producers.MRSA 112 produced the largest amount of biofilm (data not shown).All strains with antibiotypes A (except MRSA 122), B, and C produced biofilm; however, MRSA 118 strain with antibiotype E showed biofilm production.All SCCmec NT isolates produced biofilm (Table 1).

Analysis of genetic relatedness
Genetic relatedness between the MRSA isolates was analyzed by three PCR methods using complementary primers to repetitive DNA sequences in the bacterial genome.Using a cutoff value of 80% similarity, primer JB1 showed the highest discriminatory power compared with the other primers.Fourteen genotypes were discriminated by JB1, eleven by RW3A, and nine by BOX A1R.Comparing primers RW3A and JB1 using the same cutoff value, eleven genotypes were discriminated, with agreement for a few isolates (Figure 1 and Table 1).The genotypes obtained with the RW3A primer were designated 1A-1K.Seven isolates were 100% similar; thirteen clonal groups with only one isolate showing similarity of more than 80% were designated as single isolates (SIs).The genotypes obtained by JB1 were designated 2A-2N.Eleven isolates were 100% similar, and eighteen isolates were considered SIs.The genotypes obtained using BOX A1R were designated 3A-3I.Thirty-one strains were 100% similar, and thirteen isolates were considered SIs.Based on the RW3A-JB1 dendrogram, the genotypes obtained were designated by 4A-4K.Eleven isolates were 100% similar, and eighteen isolates were considered SIs.Most isolates within a given clonal group showed different phenotypes and SCCmec types.
There was no relationship between the genotyping groups based on the three primers with the SCCmec types; the presence of certain types of SCCmec was not characteristic of a specific clonal group.

Discussion
MRSA is commonly characterized as having multiple resistances to different antibiotics.
Currently, MRSA is considered the most common multidrug-resistant microorganism in hospitals [41].In Brazil, the prevalence of MRSA isolates ranges from 40% to 80% [40].A study conducted in the city of São Paulo showed the variation in susceptibility profiles, with MRSA isolates showing susceptibility to CIP (42%), CLI (55%), ERY (15%), SXT (55%), GEN (37%), RIF (78%), and TET (64%) [42].As these results are different from those of our study, the therapeutic approach in relation to the use of antimicrobials in hospitals may vary based on location (city, state, and country).
In our study, we also found high resistance to CIP, CLI, and ERY and susceptibility to LNZ.Conversely, resistance to SXT, RIF, and TET was lower.The predominant antibiotic used to treat MRSA infections at the Hospital UH/UEL is vancomycin.We observed that the majority of MRSA strains from blood (66.7%) were antibiotype D (26.7 %) or E (40,0 %) (Table 1).All isolates from urine were antibiotype O (Table 1).All MRSA strains with antibiotype D had SCCmec I, except for one that had non-typeable SCCmec.All MRSA strains with SCCmec III and IV were antibiotype A and G, respectively (Table 1).We did not find correlation of antibiotypes with patients' ages.
Although the Brazilian endemic clone (BEC) with SCCmec type III has been shown to be the clone that is spread in Brazil, this was not the SCCmec type found most frequently in our isolates; indeed, SCCmec type II was most prevalent, according to another study described by Oliveira et al. [43].Furthermore, the analysis of DNA polymorphism performed in our study showed a low genetic similarity (45%) of strain BEC9393 with most of the strains studied.Researchers from southern Brazil analyzed isolates from a hospital in Porto Alegre and found the presence of SCCmec type I, which is related to the Cordoba/Chilean clone, warning of its possible spread, replacing BEC [44].A study with blood strains collected from patients at the Hospital das Clínicas in São Paulo, Brazil, showed predominantly SCCmec type II [45].Our results and previous research show that epidemiological studies involving clonality of MRSA should be more common because new clones, including different antimicrobial resistance profiles, may arise within a period of years.
Two isolates (MRSA 410 and MRSA 308) showed SCCmec type IV, a type that is not common in healthcare-associated infections and is characteristic of CA-MRSA.In addition, these isolates showed resistance to ERY.The presence of these isolates in this hospital suggests that the spread of MRSA may occur between the two environments (community and hospital).
Most isolates showed the ability to produce biofilm, which might represent a greater difficulty in controlling MRSA infection.Six strains with mecA but different SCCmec types, I to IV (NT), all produced biofilm, showing that this important feature would be present in strains or new MRSA clones.
According to the dendrograms evaluating the similarity among the isolates, RW3A revealed the presence of 17 isolates (30.9%) with 100% similarity distributed among different clonal groups.However, the strains did not exhibit the same phenotypes, being resistant to different classes of antimicrobials according to another study [26].This result indicates the existence of identical or similar genetic profiles, but we cannot confirm that there is a clonal spread.DNA polymorphism techniques have been recommended for identifying MRSA clonality, which is extremely important in the epidemiology of the disease, and for determining the type of treatment of these infections.Using the RW3A primer, Del Velchio et al. [25] showed eight different fingerprint patterns in MRSA strains from different sources.Our study showed a higher number of DNA bands (4 to 16 bands), and some isolates did not produce a high-intensity amplicon of approximately 325 bp.This difference in DNA fingerprint profile can be due to different MRSA strains with respect to geographic distribution and antibiotic treatment.
The isolates showed a genotypic similarity profile of over 35% with the RW3A and JB1 primers, slightly higher than that reported by other researchers (20%) [26], though the previously reported similarity using BOX A1R was greater than 20%.
Genotypic analysis is an important tool for clinical practice because it provides reliable data for determining the epidemiology and pathogenesis of infectious processes.The PCR-based methods used in these studies showed good results for evaluating the similarity between S. aureus strains isolated from different locations [26,46].This method using the RW3A primer has been demonstrated to be an interesting tool for distinguishing among different genotypic profiles.In our study, the 1N clonal group (using RW3A) revealed three isolates (75%) with SCCmec type III and identical characteristics (antibiotype A and biofilm production); these three isolates showed different genotypes with JB1 and BOX.
Another isolate with SCCmec type III was designated as SI and was not able to produce biofilm on a polystyrene surface.This phenotypic characteristic was important for discriminating the SCCmec type III isolates using the RW3A primer.
The JB1 primer, which was described for Enterococcus faecium [28], exhibited desirable discriminatory power, suggesting that this primer could be used for purposes including the phylogenetic analysis of MRSA isolates.
In our study, the RW3A primer proved to be the best, and the clonal groups became more distinct with respect to genotypic and phenotypic characteristics.A previous study concluded that the correlation between antimicrobial resistance and the dissemination of clonal groups of S. aureus is an important tool during outbreaks or in endemic areas [47].
We recommend not using these three primers in combination because primer BOX A1R did not show a good discriminatory power and, in fact, interfered with the results obtained using the other primers.
Few studies have been performed using the RW3A primer for S. aureus typing.Some researchers analyzed S. aureus susceptibility and resistance to oxacillin and identified no correlation between resistance and the percentage of similarity between strains.In our study, there was no relationship between the clonal groups with particular SCCmec types and resistance profiles.
The HRM method has been introduced for Clostridium difficile ribotyping, Enterococcus species typing, and MRSA typing [48][49][50].A study developed by Chen et al. [50] using the HRM technique applied to 55 MRSA collected from Hong Kong hospital during 2011 showed 12 different types of spa in concordance with 100% of the spa type method.The authors confirmed the cost effectiveness of HRM; the method required less time to be developed and the reagent cost was one-fifth of the cost of the conventional method.Studies confirmed that HRM typing methodology could be useful for MRSA community transmission monitoring and hospital outbreak control.Our study demonstrated the practical application of this low-cost HRM method for MRSA typing on clinical isolates.
Among the 21 isolates on which were performed HRM, this methodology showed eight clonal groups and RW3A method showed nine groups, thus, both techniques did not present any clonality between MRSA isolates.However, the clonal groups obtained by the techniques were not concordant.In clonality studies in MRSA isolates, both methodologies would be interesting because they are rapid, low cost, and use the instruments available in some laboratories, as compared to MLST.
The ST generated did not represent the same groups generated by primer RW3A, as there were samples with the same ST grouped in different clonal groups (based on the primer RW3A).In two samples (MRSA 308 and MRSA 404) with the same ST5 at HRM (Table 2), there was only 40% similarity using the primer RW3A-JB1.
These results show that the RW3A primers, JB1 and BOX, are not good tools for pre-selection of samples for more discriminatory techniques such as MLST, PFGE, and spa.
For our study, the HRM technique was an important tool to verify the genotypic variability in our sample.
Although antimicrobial susceptibility tests provide information for routine surveillance, genetic typing studies with greater power of discrimination must also be performed.The correlation between clonality and antimicrobial susceptibility profile is an important tool for assessing the stage of an outbreak and characterizing the epidemiology of hospital isolates.Our results showed that the HRM technique used in our study can quickly provide important epidemiological information for MRSA with low operating costs.

Conclusions
Antimicrobial susceptibility tests provide information for routine surveillance, and genetic studies with greater discriminatory power for typing can also be performed.Correlation between the clonality of strains and antimicrobial susceptibility profiling are important tools to assess the stage of outbreaks and characterize the epidemiology of nosocomial isolates.The findings presented here indicate that the HRM technique utilized may provide important information regarding the epidemiology of MRSA, with faster results and lower operating costs.

Figure 1 .
Figure 1.Profile of similarity among isolates of methicillin-resistant Staphylococcus aureus (MRSA) using a combination of the RW3A and JB1 primers.

Table 1 .
Characteristics of methicillin-resistant Staphylococcus aureus (MRSA) isolates from patients seen at the University Hospital of Londrina-PR: SCCmec type, antibiotype, genotype, and biofilm production.

Table 1 (
continued).Characteristics of methicillin-resistant Staphylococcus aureus (MRSA) isolates from patients seen at the University Hospital of Londrina-PR: SCCmec type, antibiotype, genotype, and biofilm production.

Table 2 .
High-resolution melting (HRM) analysis profile of single-nucleotide polymorphisms of methicillin-resistant Staphylococcus aureus.
ST: sequence type.