Molecular typing of methicillin-resistant Staphylococcus aureus strains by PCR-RFLP of SPA gene: a reference laboratory perspective.
OBJECTIVE
To characterize methicillin-resistant Staphylococcus aureus (MRSA) strains by molecular typing based on polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) of spa gene and to assess the utility of spa genotyping over bacteriophage typing in the discrimination of the strains.


METHODS
Studies were undertaken on 125 MRSA strains representing the most predominant phage types and the non phage typeable strains. Strains were typed by bacteriophage typing and PCR-RFLP of spa gene. DNA sequence analysis of the amplified spa gene fragment of the representative RFLP patterns was performed using standard protocols.


RESULTS
All the strains resistant to oxacillin were found to contain mec A gene. Fifty-two per cent of these strains were typeable by the international basic set of 23 phages. Five different PCR-RFLP patterns were observed among 125 MRSA strains. Non phage typeable strains were differentiated into four PCR-RFLP patterns. Sequencing of the spa gene from the representative strains of each RFLP pattern confirmed the length of these restriction fragments due to variation in the 24 bp and the 174 bp tandem repeats. It also revealed the presence of three new spa repeat patterns.


CONCLUSIONS
The study demonstrates the importance of spa genotyping in the discrimination of MRSA strains, which were otherwise indistinguishable by bacteriophage typing. spa genotyping allowed differentiation of strains within a particular phage type. Nucleotide sequencing of isolates of different PCR-RFLP patterns indicated a correlation between the RFLP patterns of a variable number of tandem repeats and the phage type. The study provides valuable information on the epidemiological characterization of MRSA strains.
Mehndiratta
P L
PL
Department of Microbiology, National Staphylococcal Phage Typing Center, Maulana Azad Medical College, New Delhi, India. pmehndi@gmail.com
Bhalla
P
P
Ahmed
A
A
Sharma
Y D
YD
D015373:Bacterial Typing Techniques / Q000379:methods*; D001434:Bacteriophage Typing; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics*; D005838:Genotype; D006801:Humans; D055624:Methicillin-Resistant Staphylococcus aureus / Q000145:classification* / Q000235:genetics* / Q000302:isolation & purification; D017720:Molecular Epidemiology; D011110:Polymorphism, Genetic; D012150:Polymorphism, Restriction Fragment Length; D017422:Sequence Analysis, DNA; D013203:Staphylococcal Infections / Q000453:epidemiology / Q000382:microbiology*; D013205:Staphylococcal Protein A / Q000235:genetics
D004269:DNA, Bacterial; D013205:Staphylococcal Protein A