Molecular typing on 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: 1.16). 09/2005; 58(4):250-2.
Source: PubMed


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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    • "In some bacterial species, individual strains cannot be typed by PFGE because of the expression of endogenous endonucleases (Koort et al. 2002). A higher level of discrimination than that afforded by PFGE analysis would be beneficial in some studies (Willse et al. 2004) and in other cases, it would be a great advantage to be able to perform an analysis in less than several days which is typical for PFGE typing (Mitani et al. 2005). A further disadvantage of PFGE is the specialized equipment it requires, which is not available in many settings. "
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    ABSTRACT: Bacterial typing is a key technology in human and veterinary medicine, community health, consumer protection and in agricultural research. The importance of the development of epidemiological tracking tools is underlined by numerous outbreaks of diseases due to bacterial pathogens. Particularly important is tracing pathogen dissemination in 'real time' i.e. to use a fast typing technique to distinguish between clonally related (epidemic) strains and unrelated (sporadic) strains. The aim of the research was to develop a fast discriminatory molecular typing technique - double digest selective label (DDSL) for Staphylococcus aureus isolates and to compare typing data with that obtained by pulsed-field gel electrophoresis. In this new typing method, large DNA fragments are produced with a restriction enzyme commonly used for PFGE but are trimmed by a second enzyme to a size which can be separated on a conventional agarose gel within a short period of time. Selective labelling of a subset of the numerous restriction fragments gives a distinct banding pattern for each isolate. Discriminatory power obtained with DDSL calculated over two different sets was higher than that of pulsed-field gel electrophoresis. Clusters of identical isolates were further resolved to unique DDSL strains. Two combinations of restriction enzymes for DDSL technique has been proposed with approximately equal discriminatory power. It has been demonstrated that DDSL approach is a fast, discriminatory alternative to other typing techniques suitable for short-term epidemiological studies.
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    • "A similarly designed study by Mitani et al. [30] reported 8 spa and 6 coa types using HaeII restriction enzyme for coa and spa genes PCR-RFLP. Combination of these 2 techniques revealed 10 R types, where types R1–R4 were relatively frequent. "
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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.
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    • "This 24 nucleotides region is highly polymorphic with respect to the number and sequence of repeats. Diversity of X region causes variation in different protein A Staphylococcus aureus [4] [5]. Strain typing of Staphylococcus aureus is a good tool for epidemiologic "
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