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: 1.16). 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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    • "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.
    International Journal of Microbiology 05/2014; 2014:650328. DOI:10.1155/2014/650328
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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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    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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    • "). PCR- RFLP of the coagulase gene has been widely used in the clinical laboratory because of its ease and speed, and has been widely used in genotyping clinical and nonclinical S. aureus strains (Montesinos et al., 2002; Mitani et al., 2005; Shittu and Lin, 2006; Ishino et al., 2007; Moon et al., 2007; Saei et al., 2009). Moreover, macrorestriction followed by pulsed-field gel electrophoresis (PFGE) is considered the gold standard method for epidemiological typing of a variety of bacterial species including S. aureus since it is a highly discriminatory, stable and reproducible method (Faria et al., 2008). "
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    ABSTRACT: Staphylococcus aureus is one of the major causative agents of infection in all age groups following surgical wounds, skin abscesses, osteomyelitis and septicaemia. In Nigeria, it is one of the most important pathogen and a frequent micro-organism obtained from clinical samples in the microbiology laboratory. Data on clonal identities and diversity, surveillance and new approaches in the molecular epidemiology of this pathogen in Nigeria are limited. This study was conducted for a better understanding on the epidemiology of S. aureus and to enhance therapy and management of patients in Nigeria. A total of 54 S. aureus isolates identified by phenotypic methods and obtained from clinical samples and nasal samples of healthy medical personnel in Ondo and Ekiti States, South-Western Nigeria were analysed. Typing was based on antibiotic susceptibility pattern (antibiotyping), polymerase chain reaction-restriction fragment length polymorphisms (PCR-RFLPs) of the coagulase gene and pulsed field gel electrophoresis (PFGE). Of the 54 isolates, 50 were confirmed as S. aureus by PCR detection of the nuc gene. The antibiotic susceptibility testing showed that all the isolates were susceptible to fusidic acid and clindamycin, but 100% resistant to penicillin, 70% to tetracycline, 22% to erythromycin, 6% to gentamicin and 4% to ciprofloxacin. Only one isolate was confirmed to be a methicillin-resistant S. aureus (MRSA). The study established the importance of confirming phenotypic identification of S. aureus and MRSA by molecular techniques. There was a high level of agreement between the three methods in the typing of methicillin-susceptible S. aureus (MSSA). Furthermore, the recognition of a predominant MSSA clone from clinical and nasal samples indicates the possible cross-infection from medical personnel to patients. A total of nine of the 50 isolates were Panton-Valentine leukocidin (PVL) positive suggesting that they are community-associated S. aureus isolates. The study provided baseline information on the need for effective infection control measures in health-care institutions in South-Western Nigeria.
    African journal of microbiology research 01/2009; 3:962-968. · 0.54 Impact Factor
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