Genotypic and phenotypic characteristics of Methicillin-resistant Staphylococcus aureus ( MRSA ) strains , isolated on three different geography locations

Staphylococcus aureus is a major cause of hospital-acquired infections worldwide. Increased frequency of methicillin-resistant Staphylococcus aureus (MRSA) in hospitalized patients and possibility of vancomycin resistance requires rapid and reliable characterization of isolates and control of MRSA spread in hospitals. Typing of isolates helps to understand the route of a hospital pathogen spread. The aim of this study was to investigate and compare genotypic and phenotypic characteristics of MRSA samples on three different geography locations. In addition, our aim was to evaluate three different methods of MRSA typing: spa-typing, agr-typing and GenoType MRSA. We included 104 samples of MRSA, isolated in 3 different geographical locations in clinical hospitals in Zagreb, Mostar, and Heidelberg, during the period of six months. Genotyping and phenotyping were done by spa-typing, agr-typing and dipstick assay GenoType MRSA. We failed to type all our samples by spa-typing. The most common spa-type in clinical hospital Zagreb was t041, in Mostar t001, and in Heidelberg t003.We analyzed 102/104 of our samples by agr-typing method. We did not find any agr-type IV in our locations. We analyzed all our samples by the dipstick assay GenoType MRSA. All isolates in our study were MRSA strains. In Zagreb there were no positive strains to PVL gene. In Mostar we have found 5/25 positive strains to PVL gene, in Heidelberg there was 1/49. PVL positive isolates were associated with spa-type t008 and agr-type I, thus, genetically, they were community-associated MRSA (CA-MRSA). Dipstick assay GenoType MRSA has demonstrated sufficient specificity, sensibility, simple performance and low cost, so we could introduce it to work in smaller laboratories. Using this method may expedite MRSA screening, thus preventing its spread in hospitals.


INTRODUCTION
Staphylococci are among the most important causes of both hospital-and community-acquired infections worldwide.It is well known that methicillin-resistant Staphylococcus aureus (MRSA), like methicillin-sensitive Staphylococcus aureus (MSSA) could colonize or infect patients [1].MRSA strains were not found to be more virulent than MSSA strains and to cause the same spectrum of infections.S. aureus causes superficial and deep skin and soft tissue infections, bacteremia, endocarditis, osteomyelitis, pneumonia, food poisoning, toxic-shock syndrome and staphylococcal scaled skin syndrome [1][2][3][4][5].In the early 1950s, acquisition and spread of β-lactamase-producing plasmids decreased the effectiveness of penicillin for treating S.aureus infections.Methicillin, a modified penicillin, designed to resist the destructive action of the staphylococcal β-lactamase, became available for therapeutic use in 1959.However, MRSA was identified in 1960s.The resistance was a result of S. aureus' s acquiring the mecA gen, which encodes for an altered penicillin-binding protein gen (PBP2a).It was not blocked by methicillin and could replace the other PBPs, thus allowing the survival of S.aureus in the presence of methicillin [6][7][8][9].As opposed to the penicillinase gene, mecA does not reside on a plasmid but on the chromosome, embedded in a large mobile genetic element called Staphylococcal Chromosome Cassette mec or SCCmec [10,11].The presence of PBP2a means MRSA is not only resistant to methicillin but also to all β-lactam antibiotics, including synthetic penicillins, cephalosporins and carbapenems.By the early 1960s, European hospitals were reporting outbreaks of MRSA infections.In Bosnia-Herzegovina, we noted an increment of MRSA infections in the early 1990s, with the beginning of the war [12].Data from the European Antibiotic Resistance Surveillance System (EARSS) showed a rising trend of MRSA infections until 2005, with the proportion of MRSA infections varying from less than 1% in the northern to 50% in southern European countries [13].This striking difference is probably due to differences in antibiotic use or in the implementation of measures to control MRSA spread in hospitals [14].MRSA has been linked for many years-with hospital stay, homes for the elderly and infirm, and similar institutions.However, in 1990s, community-associated MRSA (CA-MRSA) has appeared, with a large number of characteristics different from previously known hospital acquired MRSA (HA-MRSA).HA-MRSA is mainly multi-resistant, and the choice of antibiotics for treating such infections is limited to glycopeptides and linezolid.Furthermore, HA-MRSA mainly causes serious infections in patients with weak immune system, after long-term hospitalization, long-term use of antibiotics, etc. [15].In previous years, strains have emerged with an intermediate susceptibility or full resistance to vancomycin (VISA and VRSA, respectively), the antibiotic that for two decades represented the drug of choice for treating MRSA infections [16].The multidrug-resistant phenotype of MRSA strains and their intrinsic β-lactam resistance make them difficult and costly to cure.
Controlling MRSA remains a primary focus of most hospital infections control programs [17][18][19][20].Knowledge of the dissemination and the molecular epidemiology of MRSA strains are required to develop effective strategies to prevent the spread of MRSA.Various molecular typing techniques have been developed to investigate the spread and evolution of MRSA.Bacterial typing method should be highly discriminatory, reproducible, standardized, widely available and inexpensive.In bacterial strain typing, both phenotyping and genotyping procedures can be used.
The most common phenotyping methods are: biotyping, serotyping, antibiotic susceptibility testing and phagotyping.Disadvantage of these commonly applied techniques may be low discriminatory power.In recent years, the quality of microbiological assays has been increasing by using molecular biology techniques such as whole cell protein profile analysis and electrophoresis of multiple-locus bacterial enzymes [21].
Staphylococcal protein A (spa) typing is based on the characterization of the spa gene, which encodes for the S.aureus specific surface protein A. The spa gene consists of different functional regions including the Fc binding region and the X-region.The polymorphic X region of the spa gene is built of a variable number of 24-bp repeating fragments.Differences occur between the fragments due to deletion, point mutation and duplication of nucleotide groups.Different repeats can be assigned an alpha-numerical code, and the order of specific repeats defines the spa type.Two systems of nomenclature are in use for spa type determination.RidomStaphType is a software enabling straightforward sequence analysis and designation of spa types via synchronization to a central server [15,[25][26][27].The discriminative power of spa typing lies between that of PFGE and MLST, and in contrast to MLST, spa typing can be used to investigate both the molecular evolution and hospital outbreaks of MRSA.The main advantage of spa typing is its simplicity, since it involves sequencing of only a single locus.
The accessory gene regulator (agr) of S. aureus is a global regulator of the staphylococcal virulence, which includes secreted virulence factors and surface proteins.The agr locus is important for virulence in a variety of animal models of infection, and has been assumed by inference to have a major role in human infection.S.aureus strains have been divided into four agr specificity groups.
The GenoType MRSA detects the mecA gene and, in addition, a highly specific sequence for S. aureus by polymerase chain reaction (PCR) and reverse hybridization.Molecular genetic testing with the GenoType MRSA kit needs much less time than conventional microbiological methods.Therefore genetic testing provides not only a considerable advantage with respect to reliability but also to speed [28].
The aim of this study was to investigate and compare genotypic and phenotypic characteristics of MRSA strains in three different geography location and to evaluate the efficacy of three different methods of MRSA typing: spa-typing, agr-typing and GenoType MRSA.

Samples
The study was carried out at 104 samples of MRSA, collected during the period of six months in three different locations in Europe: Clinical Hospital Center Zagreb, Croatia (30 samples), University Clinical Hospital Mostar, Bosnia-Herzegovina (25 samples), and University Clinical Heidelberg, Germany (49 samples).These isolates were taken from wound swabs, blood cultures, respiratory tract specimens, urine samples and surveillance cultures.MRSA isolates were collected and stored at -20 o C until analyzed.

Methods
The strains were identified with current phenotypic methods.After 24 hours of incubation at 37 o C the colonies of S.aureus on 5% blood agar were 1 to 3 mm in diameter, pigmented yellow, smooth, and convex.Identification of S.aureus was carried out by detection of DNase.All strains were DNase positive.The antibiotic susceptibility testing was performed by the disk-diffusion method, according to Clinical and Laboratory Standard Institute (CLSI) guidelines.We tested penicillin, erythromycin, azithromycin, gentamycin, amikacin, trimethoprim-sulfamethoxazole, clindamycin, rifampin, cefoxitin, linezolid, teicoplanin and vancomycin.Antimicrobial susceptibility testing was confirmed with VITEK 2 Compact (Bio Mérieux, France).

DNA extraction
DNA was extracted from samples using the InstaGene Matrix (BioRad, Austria), according to the manufacturer´s protocol.Three colonies from overnight culture of S.aureus were suspended in 500 μL of high performance liquid chromatography (HPLC) grade water and added to 100 μL InstaGene Matrix and vortexed, followed by heating at 56 o C for 20 minutes.The samples were vortexed again and heated at 100 o C for 8 minutes and then centrifuged to pellet the matrix.Aliquots of 80 μL were used as templates for PCR.

DNA amplification
The spa typing method is based on sequencing of the polymorphic X region of the protein A gene (spa), present in all strains of S. aureus.The X region is constituted of a variable number of 24-bp repeats flanked by well-conserved regions.This single-locus sequence-based typing method combines a number of technical advantages, such as rapidity, reproducibility, and portability.Moreover, due to its repeat structure, the spa locus simultaneously indexes micro-and macro variations, enabling the use of spa typing in both local and global epidemiological studies.These studies are facilitated by the establishment of standardized spa type nomenclature and Internet shared databases.
The X region of the spa gene was amplified by PCR with primers spa-1113f (5´-TAA AGA CGA TCC TTC GGT GAG C -3´) and spa-1514r (5´-CAG CAG TAG TGC CGT TTGCTT -3´).The PCR amplification was performed using Perking Elmer 9700 thermal cycler(Norwalk, CT, USA) with an initial activation step at 80 o C for 5 minutes, followed by 30 cycles of denaturation at 94 o Cfor 45 seconds, annealing at 60 o C for 45 second extension at 72 o C for 90 seconds, followed by final extension step at 72 o C for 10 min.

Detection of products
The amplicons of PCR reactions were visualized using UV light box, after the electrophoresis on a 2% agarose gel with ethidium bromide.

DNA purification
For spa typing the amplified PCR products were purified using a common purification kit (New England Biolabs GmbH, Frankfurt-Hoechst, Germany and Amersham Pharmacia Biotech).Briefly, 5 μL of the PCR product was incubated with 1U of each enzyme Exonuclease I and Shrimp Alkaline Phosphatase, at 37°C for 30 minutes.Then the enzymes were inactivated at 80°C for 15 minutes and the PCR products were stored at 4°C.

PCR for sequencing
At least 30 ng of the above purified PCR product was used for PCR sequencing.ABI Big Dye Terminator Ready Reaction kit Version 3.1 (PE Applied Biosystems, CA, USA) was used under the following conditions: splitting chains at 95 o C for 3-4 minutes, followed by 25 cycles of annealing at 60 o C for 30 seconds, building of nucleotides at 60 o C for 4 minutes, and splitting chains at 95 o C for 30 seconds, and extension at 60 o C for 7 minutes.Templates were purified for sequencing by Qiagen Spin Kit DyeEx (Qiagen GmBH, Hilden, Germany).

DNA sequencing
DNA sequences were obtained with an ABI 377 sequencer (Applied Biosystems, Foster City, Calif.).
Spa types were determined with the RidomStaphType software (Ridom GmbH, Wurzburg, Germany).

DNA extraction
DNA extraction was made as described above.

DNA amplification
Agr specificity groups were identified by PCR amplification of the hypervariable domain of the agr locus using oligonucleotide primers specific for each of the four major specificity groups.A forward primer, pan-agr (5'-ATGCACATGGTGCACATGC-3'), corresponding to conserved sequences from the agrB gene, was used in all reactions(primer sequences were obtained from GenBank accession numbers X52543, AF001782, AF001783, and AF288215).Four reverse primers, each specific for amplification of a single agr group based on agrD or agrC gene nucleotide polymorphism, were as follows: 1. agr I, 5'-GTCACAAGTACTATAAGCTGCGAT-3' (in the agrD gene) 2. agr II, 5'-GTATTACTAATTGAAAAGTGC CATAGC-3' (in the agrC gene) 3.
agr IV, 5'-CGATAATGCCGTAATAC CCG-3' (in the agrC gene) The PCR assay was performed in 50 μL of reaction mixture containing 5 μL KCl buffer, 8 μL MgCl2, 10 μL dNTP (dATP, dCTP, dGTP, dTTP), 2 μL of Taq polymerase, 20 pmol of each primer, and 10 μL of isolated DNA.The reaction mixtures were placed in a Perking Elmer thermal cycler (Norwalk, CT, USA).The thermal profile involved an initial denaturation step at 94°C for 5 min followed by 30 cycles of denaturation at 94°C for 1 min, annealing at 55°C for 1 min, and elongation at 72°C for 1 min.The cycling was followed by a final extension step at 72°C for 5 min.S. aureus strains RN6390 (agr group I), RN6607 (agr group II), RN8465 (agr group III), RN4550 (agr group IV), and RN6911 (agr negative) were used as controls for agr group identification, and were kindly provided by Prof Wolfgang Witte PhD.Aliquots of amplified samples were analyzed by electrophoresis on a 1% agarose gel and stained with ethidium bromide.

GenoType MRSA
GenoType MRSA is newly available molecular genetic test kit (Hain Lifescience GmbH, Nehren, Germany), which detects the mecA gene and, in addition, a highly specific sequence for S. aureus by polymerase chain reaction (PCR) and reverse hybridization [28].We have analyzed all our strains by the dipstick assay GenoType MRSA.

DNA extraction
Five overnight S. aureus colonies were re-suspended in 150 μLof water.Bacterial DNA was released by incubation of the solution for 15 min at 95°C, followed by incubation in an ultrasonic bath for 15 min, and spun down for 5 min at maximum speed.Afterwards, we used 5 μL of supernatant for PCR.

DNA amplification
The PCR was performed using a hot start Taq polymerase (HotStartTaq, Quiagen, Germany).The amplification mix contained 35μL PNM (biotin-labeled primers and dNTP), 5 μL polymerase incubation buffer, 2 μL 25 mM MgCl 2 , 1U polymerase, 2 μL distilled water and 5 μL DNA solution.The amplification was carried out in a Perking Elmer 9700 thermal cycler (Norwalk, CT, USA).The sensitivity of amplification and hybridization was monitored using an internal control.

Hybridisation
PCR products (20 μL) were mixed for 5 min with 20 μL of denaturing reagent (provided with the kit) at room temperature in separate troughs of a plastic tray.After addition of 1 ml of pre-warmed hybridization buffer, the membrane strips in the kit were added to every trough.Hybridization was at 45°C for 30 min, followed by two washing steps at 45°C for 30 min with 1 ml of pre-warmed stringent wash solution.For colorimetric detection of hybridized amplicons, streptavidin-conjugated alkaline phosphatase and the appropriate substrate were added.After final washing, the strips were airdried and fixed on a data sheet.

Statistical methods
Statistical analysis of the data was performed using SPSS for Windows (version 13.0, SPSS inc, Chicago, Illinois, USA) and Microsoft Excel (version 11, Microsoft Corporation, Redmond, WA, USA).Fisher' s exact test and χ 2 test, were used to compare categorical variables between groups.The p values < 0.05 were considered statistically significant.

Antimicrobial susceptibility
All MRSA isolates were resistant to the tested β-lactam antibiotics, i.e. penicillin, oxacillin and cefoxitin, and all isolates were susceptible to linezolid, teicoplanin and vancomycin.

Spa-typing
As described in introduction, the polymorphic X region of the spa gene is built of a variable number of 24-bp repeating fragments.According to RidomSpa Server, 45 of 104 analyzed strains of MRSA did not belong to any known spa-type.Using spa-typing method, we have successfully analyzed 59/104 (56.7%) of our samples.There were 11 different types.The most common spa-type was t001(χ 2 =70.586; df=10; p<0.001)(Figure 1A).
MRSA strain frequency were significantly different according to agr-types, depending on the location of research (Monte Carlo 2-sided; p=0.011).In CHC Zagreb, we have found exclusively agr-type II 29/29 (100%), in UCH Mostar agr-type I and agr-type II and in UCH Heidelberg agr-type I, agr-type II and agr-type III (Figure 2B).

Distribution of agr-types according to type of sample
All isolates from clinical hospital Zagreb belonged to agr-type II.Most of the isolates were from the wound swab (Table 1).There was no significant difference in the agr-type depending of the source of the samples in the Mostar (Monte Carlo 2-sided; p=0.556) (Table 1).
There was no significant difference in the agr-type depending of the source of the samples in the Heidelberg (Monte Carlo 2-sided; p=0.381) (Table 1).Looking to the isolates from all 3 locations, we did not see any significant difference in the agr-types frequencies according to the source of the sample (Monte Carlo 2-sided; p=0.645).Spa-types varied significantly depending on the agr-types (Monte Carlo 2-sided; p<0.001).spa-type t008 (77.8%) predominated in agr-type I. spa-types t001 (35.4%) and t003 (31.3%)predominated in agr-type II.

GenoType MRSA
We analyzed all our samples by the dipstick assay GenoType MRSA.All isolates were positive to mecA gene.In clinical hospital Zagreb there were no positive strains to PVL gene, and in Heidelberg there was 1/49.In Mostar we have found 5/25 positive strains to PVL gene which is significantly more PVL-positive strains compared to the other two locations (Monte Carlo 2-sided; p=0.011) (Figure 3 and Table 2).

DISSCUSION
S. aureus is a major nosocomial pathogen that causes a range of diseases, including endocarditis, osteomyelitis, pneumonia, toxic-shock syndrome, food poisoning, carbuncles, boils and infection of surgical wounds.Increased frequency of MRSA or multidrug-resistant phenotype of MRSA strains and their intrinsic beta-lactam resistance, make them difficult and costly to treat.
Bacterial strain typing, or subtyping, has become an important clinical tool to investigate suspected outbreaks and to evaluate nosocomial transmission [29].Numerous techniques are available to differentiate MRSA isolates.Historically, isolates were distinguished by phenotyping methods, however, many S.aureus isolates cannot be typed using this method.Genotyping methods have significant advantages [30].
In this study were examined 104 samples of MRSA, collected during the period of six months on three different locations in Europe.These isolates were taken from wound swabs, blood cultures, respiratory tract specimens, urine samples and surveillance cultures.The samples were obtained from patients in hospitals (97, 93.3%) and outpatient setting (7, 6.7%).
Sensitivity to antibiotics is a phenotypic method, which  is performed in all clinical microbiology laboratories.Diskdiffusion method is carefully standardized and reproducible, both within the laboratory and between different laboratories.But this method is limited in most of the epidemiological investigations, because genetically and epidemiologically unrelated strains can show the same pattern of sensitivity and resistance.However, despite these limitations, the routine preparation of susceptibility testing can detect antimicrobial resistance, which is very important and it is often early warning of a problem.We characterized MRSA strains by using different molecular typing tools.Using spa-typing method, developed by Frenay et al. [25], we successfully analyzed 56.7% of our samples.There were 11 different spa-types with the most common being t001.In Zagreb the most common spa-type was t041 (64.3%), in Mostar t001 (64.7%) and in Heidelberg the most common type was t003 (53.6%).According to RidomSpaServer the spa-type t041, which was dominant in Zagreb (64.3%), is globally much less present (0.4%).This type is described as a Southern German MRSA.The same strain was found in Mostar in 11.8% of samples, while it was not found in Heidelberg.
In clinical hospital Heidelberg the most common type was t003 (53.6%).According to RidomSpaServer, it is globally the most represented type (10.9%).This strain was not found in Zagreb and Mostar.The high frequency of t041 in clinical hospital Zagreb and t001 in Mostar restricts the usefulness of spa-typing for local investigations.According to Vindel et al. most common spa-types in Spain, were t067 and t002, which is in contrast to relatively low frequency of these types in other European countries [31].Most common agr-type was agr-type II (83.3%), then agrtype I (15.1%), agr-type III (1%), while we did not find samples with agr-type IV.In Zagreb, agr-type II 29/29 (100%) was the only type found, in Mostar agr-type I (20%) and agr-type II (80%) and in Heidelberg agr-type I (22.9%), agr-type II (75%) and agr-type III (2.1%).Our hypothesis was that we could classify the samples in 4 agr-types, and to investigate their genetic background and possible relation between agr-type and the capacity to induce a specific disease [32].While our results do not show a direct role of the agr-type in the type of human disease caused by MRSA, the higher prevalence of agr-type II in our samples could suggest that agr-type II is associated with nosocomial MRSA infections [33].
With the GenoType MRSA we expected to get sufficiently sensitive, specific, fast and low cost method for MRSA typing, which could be introduced to smaller microbiological laboratories.A PCR based test was developed for the detection of mecA in staphylococci.A various methods of mecA detections were described [34].Some of them use gel-electrophoresis for visualization of amplifications products.However, these techniques are time consuming and require expensive equipment, and they are not acceptable in a smaller microbiological laboratories for daily use.All isolates in present study were positive to mecA gene.In Zagreb there were no positive strains to PVL gene.In Mostar we have found 5/25 (20%) positive strains to PVL gene, and in Heidelberg there was 1/49 (2%).
According to definition of community acquired MRSA (CA-MRSA), i.e. strains isolated in an outpatient setting, or from patients within 48h of hospital admission [35][36][37] we found that neither of 5 PVL positive strains from Mostar were not CA-MRSA.
Described strains were resistant to penicillin, macrolide, and ciprofloxacin, and were susceptible to all other tested antibiotics.One of that strain was susceptible to ciprofloxacin.All five strains have belonged to spa-type 008 and agr-type I.
One PVL positive strain from Heidelberg, according to medical documentation, was CA-MRSA.This strain was resistant to penicillin, macrolides and clindamycin, but was susceptible to all other tested antibiotics.The strain belonged to spa-type 008, and agr-type I, the one described by Witte et al. as the first case of CA-MRSA in Germany 2005 [38].Blanco et al. conducted a study of PVL positive strains, which all belonged to spa-type 008, and agr-type I. Numerous studies show that the strains of CA-MRSA are expanded all over the world, although their prevalence varies from one area to another [39].In the United States, CA-MRSA clone designated as USA 300 has become the most widespread [40].This strain of recently started to cause the outbreaks in neonatal wards [41].
The prevalence of infections caused by CA-MRSA is a lower in Europe compared to USA, but recently and increasing trend is observed.Typical CA-MRSA is sensitive to many non-β-lactam antibiotics.However, increased use of these antimicrobials could lead to emergence of new multidrug resistant clones [42].There are no sufficient data about the prevalence of CA-MRSA in Bosnia-Herzegovina.There is a wide range of methods for genotyping of MRSA strains for epidemiological research.Molecular testing will continue to be an essential tool and the testing has proven to be cost-effective and medically justified.

CONCLUSION
Antimicrobial susceptibility, as a phenotyping method, is a simple one to perform and interpret.However, many S.aureus strains cannot be typified using this method.Our results do not show a direct role for the agr-type in the type of human disease caused by MRSA.Higher prevalence of agr-type II in our samples could suggest that agr-type II is associated with nosocomial MRSA infections.Dipstick assay GenoType MRSA has demonstrated sufficient specificity and sensibility, simplicity of performance and low cost, so it could be introduced into small microbiological laboratories for expediting the MRSA screening and preventing its spread.

FIGURE 2 .FIGURE 1 .
FIGURE 2. MRSA agr-types collected from Mostar (Bosnia and Herzegovina), Zagreb (Croatia) and Heilderberg (Germany) hospitals.(A) Total number of isolates according to MRSA strain.(B) Number of samples collected in each institution according to MRSA strain.B A

FIGURE 3 .
FIGURE 3. Total number and PVL-positive strains from all three locations.

TABLE 1 .
Agr-types according to samples from CHC Zagreb, UCH Mostar and UCH Heidelberg

TABLE 2 .
Genotypic characteristics of Methicillin-resistant Staphylococcus aureus (MRSA) strains, isolated on three different geography locations: Mostar, Zagreb and Heidelberg