Phenotypic and molecular characterization of HA-MRSA in Taif hospitals , Saudi Arabia

Introduction: Methicillin-resistant S. aureus (MRSA) is one of the most important organisms causing hospital-acquired infections worldwide. Molecular analysis of MRSA strains from Taif, Saudi Arabia, had not been previously done. Phenotypic and molecular characteristics of MRSA isolated from Taif hospitals were investigated. Methodology: This study involved S. aureus strains isolated from different clinical samples from Taif hospitals. MRSA strains were identified and antimicrobial susceptibility profiles were determined. Multiplex polymerase chain reaction (PCR) was used to identify S. aureus-specific sequence, mecA genes, and type of staphylococcal cassette chromosome mec (SCCmec). MRSA strains were typed using coagulase gene polymorphism. Results: In total, 390 strains of S. aureus were isolated, and 58 MRSA strains – 40 hospital-acquired MRSA (HA-MRSA) and 18 community-acquired MRSA (CA-MRSA) – were detected. HA-MRSA strains included three SCCmec types, while CA-MRSA strains included two SCCmec types. PCR amplification and restriction of the coagulase gene of the 58 MRSA isolates showed nine different patterns, while three strains were non-typable. HA-MRSA strains showed seven distinct restriction fragment length polymorphism (RFLP) patterns; the most frequent was pattern 2 (15 isolates), followed by patterns 1 and 4 (5 isolates each). CA-MRSA showed five RFLP patterns; the most frequent was pattern 3 (7 isolates) followed by pattern 8 (6 isolates). Conclusions: HA-MRSA strains were more common than CA-MRSA strains. SCCmec typing and coagulase gene polymorphism analysis may be useful methods for studying clonal relatedness of isolates and for discriminating between HA-MRSA and CA-MRSA.


Introduction
Healthcare-associated infections (HAIs) are considered among the most prevalent complications of hospital care [1].Staphylococcus aureus (S. aureus) is responsible for a wide spectrum of infections ranging from superficial skin infections to fatal invasive infectious diseases including pneumonia, septicemia, and endocarditis [2].Methicillin-resistant S. aureus (MRSA) was first reported in the early 1960s and has become a major nosocomial pathogen worldwide [3].In the Gulf region, MRSA has been considered to be one of the most prevalent pathogens by Gulf Corporation Council (GCC) countries in the last two decades [4].
Methicillin resistance results from introduction of staphylococcal cassette chromosome mec (SCCmec) into methicillin-susceptible S. aureus strains.SCCmec is a genomic island that contains the antibiotic resistance gene mecA [5].SCCmec also contains ccrA and ccrB; both genes encode recombinases that mediate the site-specific integration and excision of the SCCmec element from the S. aureus chromosome [6,7].The mecA gene codes for a variant type penicillin-binding protein (PBP2a), which has a lower affinity for the β-lactam antibiotics because its active site does not bind methicillin or other β-lactam antibiotics.Therefore, the transpeptidation reaction required for peptidoglycan cross-linking will continue, enabling cell wall synthesis [8].Seven main types of SCCmec (type I to VII) are recognized.Types IV, V, VI, and VII code for β-lactam antibiotic resistance only, while SCCmec I, II, and III cause resistance to multiple classes of antibiotics, due to additional integrated drug resistance genes [9].
There are two major strains of MRSA: hospitalacquired MRSA (HA-MRSA) and communityacquired MRSA (CA-MRSA).They have been proven to be genetically distinct with respect to the SCCmec type; most HA-MRSA strains carry one of three types of SCCmec (types I, II, or III).On the other hand, CA-MRSA strains carry mainly SCCmec type IV and occasionally type V [10].
In Saudi Arabia, few studies have been conducted to investigate the epidemiology of MRSA in the community or in hospitals.In 1991, a study showed that Saudi and British strains differed in their sensitivity patterns to antibiotics [11].In Riyadh, it was found that overcrowding of patients and inadequate infection control measures were the major risk factors for MRSA acquisition [12].In Abha (south region of Saudi Arabia), MRSA accounted for 5.1% of hospital and 18.3% of non-hospital isolates [13].To our knowledge, molecular analysis of MRSA strains in Taif area of Saudi Arabia has not been previously done.This study was conducted to investigate the phenotypic and molecular characteristics of MRSA isolates in Taif region, Saudi Arabia, and to compare CA-MRSA and HA-MRSA strains.

Specimen collection and phenotypic testing
This study was conducted on MRSA strains collected form Taif hospitals between January 2013 and February 2014.Demographic, clinical, and laboratory data were collected after written consent was obtained.The clinical specimens of MRSA isolates included sputum, pus, pharyngeal swabs, urine, blood, and peritoneal fluid.The study proposal was approved by the Faculty of Applied Medical Sciences Ethics Committee of Taif University.

Identification and antibiogram of S. aureus
Initial screening and identification of S. aureus were performed according to the standard laboratory protocols.Isolates were further confirmed by detection of staphylococcal-specific genes by polymerase chain reaction (PCR).Antibiotic susceptibility profiles were determined and interpreted according to the guidelines of the Clinical and Laboratory Standards Institute (CLSI).
Methicillin resistance was tested by standard oxacillin salt agar screening plate procedure and cefoxitin susceptibility as indicated by the CLSI.Methicillin-resistant and methicillin-sensitive strains were included as controls [14].Methicillin resistance was further confirmed by the detection of the mecA gene by the PCR method.

DNA extraction
Bacterial DNA was extracted by PrepMan Ultra Sample Preparation Reagent (Applied Biosystems, Foster city, USA) according to the manufacturer's instructions.Briefly, 1 mL of overnight lysogeny LB broth culture was centrifuged for 2 minutes.The pellet was re-suspended in phosphate buffer saline, then centrifuged again and the supernatant was discarded.PrepMan Ultra Sample Preparation Reagent (100 μL) was added.The tubes were placed in a heat block at 100°C for 10 minutes.The tubes were allowed to cool to room temperature and centrifuged at the highest speed for 2 minutes.Then, 50 μL of the supernatant was stored at -20°C and 5 μL was used for each PCR assay.

Staphylococcal cassette chromosome mec typing
SCCmec typing was done by the multiplex PCR method [16].The multiplex PCR included eight loci (A through H) selected on the basis of the previously described mec element sequences.The multiplex PCR was performed using the GeneAmp PCR kit (Applied Biosystems).

PCR-restriction fragment length polymorphism (RFLP) typing of MRSA
MRSA strains were typed using coagulase gene polymorphism.The 3' end region of the coagulase gene was amplified by PCR as previously described by Lawrence et al. [17].PCR amplification of the variable region of the coagulase gene was performed using primers COAG2-5'CGAGACCAAGA TTCAACAAG3' and COAG3-5'AAAGAAAACCACTCACATCA3'.
In this study, the SCCmec types correlated well with the major antibiotic susceptibility patterns.Although previous studies have shown that SCCmec types IV and V, which are CA-MRSA strains, were generally susceptible to non β-lactam antibiotics and more susceptible to antibiotics than were HA-MRSA strains [3,20].Moreover, our results demonstrated that SCCmec type IV (2 isolates) and SCCmec type V (3 isolates) were resistant to many non-β-lactam antibiotics.This finding was noted in previous studies, suggesting that SCCmec types IV and V strains can acquire resistance to non-β-lactam antibiotics in order to tolerate hospital environments and excessive antibiotic exposure [27].
Pulsed-field gel electrophoresis (PFGE) is considered the most discriminatory and reliable method of typing.However, it is more complex and time consuming.Furthermore, it was not available in our laboratory.PCR-RFLP typing of the coagulase gene (coa) can be used for S. aureus typing on the basis of DNA variation within the 3' end coding region of the gene [28].Since the isolation of the staphylococcal coa gene in 1987, AluI and, to a lesser extent, Hue,III-RFLP digestion of the coa gene has been extensively used for differentiation of MRSA strains with excellent correlation between RFLP patterns and multi-locus enzyme electrophoresis [2].PCR amplification and restriction of the coa gene of the studied 58 MRSA isolates generated nine different patterns.Four RFLP patterns (2,4,7,9) were detected only in HA-MRSA, and two RFLP patterns (3,5) were detected only in CA-MRSA, while other RFLP patterns (1,6,8) were shared by both types of MRSA.Our results are in accordance with those of Kobayashi et al. [29], who found that MRSA and MSSA were classified into 6 and 12 RFLP patterns, respectively, with 5 patterns detected frequently in both MRSA and MSSA.Walker et al. [18] showed that AluI digestion of the coa gene PCR products of 356 MRSA strains yielded 13 different RFLP patterns.In contrast to our results, Lawrence et al. [17] isolated MRSA strains from various hospitals and showed that the strains were closely related and that 91.8% of the isolated strains had a unique RFLP pattern when analyzed by coagulase gene typing.

Conclusions
In conclusion, HA-MRSA strains were more common than CA-MRSA strains in Taif.Coagulase gene polymorphism requires only small quantities of DNA, and isolates can be compared by both the number of PCR products and the size of their AluI restriction fragments.MRSA RFLP analysis yielded nine distinct types, and HA-MRSA and CA-MRSA were discriminated to a great extent.Coagulase gene polymorphism is an easy and accurate method for studying clonal relatedness of isolates.

Table 1 .
Clinical sources of the isolated MRSA andMSSA strains

Table 2 .
Characterization of HA-MRSA strains, SCCmec type, PCR-RFLP of coa and mecA genes