Molecular typing of methicillin-resistant Staphylococcus aureus by PCR-RFLP and its usefulness in an epidemiological study of an outbreak.

Central Clinical Laboratory, Nara Medical University, Nara 634-8522, Japan.
Japanese journal of infectious diseases (Impact Factor: 1.2). 09/2005; 58(4):250-2.
Source: PubMed

ABSTRACT A new convenient molecular typing method, simultaneous polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) analysis, for three different genes of methicillin-resistant Staphylococcus aureus (MRSA) was evaluated using 35 isolates of MRSA and comparing results with those previously reported for sequencing-based spa typing. Twenty-nine isolates of the most frequent protein A (spa) type were discriminated into 6 different types by PCR-RFLP. In contrast, spa typing could discriminate only 1 of the 19 most frequent PCR-RFLP-type isolates. The discriminatory powers of the two methods were equal for the other isolates. These results suggest that PCR-RFLP has the advantages of both relative easiness and greater discriminatory power than spa typing. We also report the case of a suspected outbreak in which PCR-RFLP was sufficient for ruling out the possibility of an outbreak. Thus, PCR-RFLP is preferable as a preliminary screening method for epidemiological studies of nosocomial infection caused by MRSA.

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    ABSTRACT: Protein A of Staphylococcus aureus is a pathogenic factor whose encoding gene, spa, shows a variation in length in different strains. In this study the spa gene variation in S. aureus isolated from healthy carriers and patients was studied, We also compared this variation among MRSA with MSSA strains. 208 strains of Staphylococcus aureus which we were isolated from Gorgan, north of Iran were studied, 121 cases from patients and 87 cases from healthy carriers, 59 out of them were MRSA and 149 MSSA. Samples DNA were extracted and amplified by specific primer of spa gene. In 4 (3.8%) strains of them no spa gene was detected, and 10.6% had a dual band (1200 and 1400 bp). In strains with one band, the length of spa gene differed from 1150 to 1500 bp. The most prevalent length was 1350–1400 bp (37%). The frequencies of short spa bands (1150–1200 bp) in patients strains were significantly higher. In 4 (3.8%) strains of them no spa gene was detected, and 10.6% had a dual band (1200 and 1400 bp). In strains with one band, the length of spa gene differed from 1150 to 1500 bp. The most prevalent length was 1350–1400 bp (37%). The frequencies of short spa bands (1150–1200 bp) in patients strains were significantly higher. The spa gene length of 1350–1400 bp in MSSA was more than in MRSA strains (P < .05). The average length of spa in isolated strains from urinary tract infections was more than others. It is concluded that the length of spa gene depends either on resistance to Methicillin or the source of S. aureus isolation.
    International Journal of Microbiology 08/2010; 2010. DOI:10.1155/2010/351397
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    ABSTRACT: Increased frequency of methicillin-resistant Staphylococcus aureus (MRSA) in hospitalized patients requires rapid and reliable characterization of isolates for control of MRSA spread in hospitals. This study evaluated polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) as a molecular typing technique for MRSA strains on the basis of protein A (spa) and coagulase (coa) gene polymorphisms to verify their ability in assessing the relatedness of isolates. Seventy-five MRSA isolates, from different ICUs of Alexandria University Main Hospital, were characterized using antibiotyping and PCR-RFLP analysis of coa and spa genes. Thirty-two antibiotypes were identified. coa gene PCR generated 3 types and 10 subtypes of band patterns. HaeIII restriction digestion of amplified coa gene products produced 5 major banding patterns and 12 subtypes. spa gene PCR products generated 4 major and 11 minor types, and their HaeII restriction digestion showed 5 major and 12 minor banding patterns. The combined coa and spa RFLP patterns generated 22 combined R types. Typing using coa PCR and PCR-RFLP had the same discriminatory index (DI) value (0.64), which was comparable to that of both spa PCR and PCR-RFLP techniques (0.68). The combined grouping increased the DI value to 0.836. The current study revealed that testing for multiple gene polymorphisms is more useful for local epidemiologic purposes.
    International Journal of Microbiology 05/2014; 2014:650328. DOI:10.1155/2014/650328
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    ABSTRACT: In a comparative study, isolates of Staphylococcus aureus from bovine mastitis cases with known coa and aroA types were analyzed by molecular typing based on polymerase chain reaction and restriction enzyme analysis (REA) of protein A-encoding gene (spa) for assessment of its utility over coa and aroA typing in discrimination of the isolates. Fifty-eight isolates of S. aureus from nine dairy herds in two Iranian provinces were typed based on polymorphism characterizing the gene encoding for the X region of protein A (spa). Five differently sized amplicons of approximately 1,200 bp to 1,410 bp were observed. Spa gene REAs produced a total of eight distinct patterns, designated as S1–S8, after digestion with restriction endonuclease Hin6I. For the spa gene, the lack of amplification was also considered a distinct genotype (S9). The majority of isolates were classified into spa types S2 (24.14%) and S6 (24.14%). This study also showed that genetic analysis of the repeat region of protein A might help to understand the distribution of prevalent S. aureus clones among bovine mastitis isolates. Interestingly, based on spa, coa, and aroA typing, some isolates which were identified as dominant types by one method were classified as rare types by another and vice versa. Finally, spa typing has 62.1% concordance with coa typing from the standpoint of the assignment of the isolates to predominant and rare lineages. This study also demonstrates the importance of spa genotyping in the discrimination of S. aureus isolates, which were otherwise indistinguishable by coa and aroA gene typing.
    Comparative Clinical Pathology 10/2010; DOI:10.1007/s00580-010-1150-y


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