Evolution, Molecular ; Methicillin Resistance ; Staphylococcus aureus ; classification ; drug effects ; genetics ; isolation & purification

BACKGROUND: Multiple-locus variable-number tandem-repeat fingerprinting (MLVF) is based on multiplex PCR, utilizing variable number tandem repeat. Our goal was to compare the performance of MLVF in distinguishing clinical Staphylococcus aureus isolates with that of pulsed-field gel electrophoresis (PFGE), which has traditionally been the gold standard. METHODS: Sixty-three clinically significant S. aureus isolates were tested using both PFGE and MLVF. Multiplex PCR for MLVF was performed using PCR primers for clfA, clfB, sdrCDE, sspA, and spa. PFGE was performed with genomic DNA fragments generated by SmaI endonuclease digestion. Banding patterns of MLVF or PFGE were analyzed using InfoQuestFP software. RESULTS: The hands-on time of our modified method was about 3 h, on average, for each of 18 isolates. PFGE (80% cutoff) or MLVF (75% cutoff) separated all of the 63 isolates into 13 and 12 types, respectively. Three types generated by PFGE were identical to those generated by MLVF. PFGE and MLVF yielded similar Simpson's diversity indices, indicating similar discriminatory power. The overall concordance between PFGE and MLVF was low, as represented by adjusted Rand indices (0.266-0.278). PFGE predicted MLVF type better than MLVF predicted PFGE type, as reflected by Wallace coefficients (PFGE cutoff 80% vs. MLVF cutoff 75%, 0.389 vs. 0.233). Analysis of the relationship between a pair of isolates showed 91.0% concordance between the PFGE (80% cutoff) and MLVF (75% cutoff). CONCLUSIONS: Our simple, low-cost, modified MLVF protocol can effectively discriminate between S. aureus clinical isolates. MLVF can replace PFGE for the hospital infection control of S. aureus.
Bacterial Typing Techniques/*methods ; *DNA Fingerprinting ; DNA, Bacterial/analysis ; *Electrophoresis, Gel, Pulsed-Field ; Genotype ; Humans ; Methicillin-Resistant Staphylococcus aureus/classification/genetics/isolation & purification ; Multiplex Polymerase Chain Reaction ; Staphylococcal Infections/*microbiology ; Staphylococcus aureus/*classification/*genetics/isolation & purification

One hundred seven isolates of Staphylococcus aureus from bovine mastitis were investigated for colony morphology in serum-soft agar (SSA), autoagglutination in salt, and capsular serotype. Capsular polysaccharide (CP) was purified and quantified from the extracts of clinical isolates. Overall, 89 isolates (83.2%) were diffuse in the SSA, without any difference in the proportion of diffuse colony between type 5 and type 8 strains. Some strains exhibited compact colonies in the SSA and expressed CP as determined by an enzyme-linked immunosorbent assay, indicating that compact morphology does not exclude encapsulation. The majority of the strains (11/12) showed autoagglutination in the salt aggregation test. The serotype 336 accounted for 46.7% of the isolates followed by serotype 5 (12.1%) and serotype 8 (12.1%). Particularly, twenty-six (24.3%) isolates reacted with two serotypes; 7 for type 8/336 and 19 for type 5/336. Five isolates (4.7%) were nontypeable with monoclonal antibodies specific for CP serotype 5, 8, or 336. The CP concentration in culture supernatants varied with the serotypes, and the total amount of CP produced by cells grown in a liquid medium was much less than that produced by cells grown on a solid medium. The Western blotting indicated that the CP bands of S. aureus serotype 5 and 8 were ranged in the molecular mass of 58-84 kilodalton (kDa), with additional bands in the region of approximately >or= 48 or Agglutination Tests ; Animals ; Bacterial Vaccines/administration & dosage ; Cattle ; Cattle Diseases/immunology/microbiology ; Female ; Mastitis, Bovine/*microbiology ; Polysaccharides, Bacterial/*classification/isolation & purification ; Serotyping/methods/veterinary ; Staphylococcal Infections/immunology/*veterinary ; Staphylococcus aureus/*classification/*immunology/isolation & purification

OBJECTIVETo study the molecular types of Staphylococcus aureus isolated from a severe food-poisoning and to trace the possible strains.

METHODSReal-time PCR was applied to detect nuc gene as a specific marker for S. aureus, mecA gene encoding methicillin resistance and 5 other genes encoding staphylococcal enterotoxins (sea, seb, see, sed, see). Isolates were also performed with 16S rRNA oligonucleotide sequence analyzing by DNAStar MegAlign 5.0 software and pulse-field gel electrophoresis (PFGE) by BioNumerics Version 4.0 software.

RESULTSThe nuc gene was detected from the 10 isolated strains, sea and seb genes were detected from 7 strains. There were 4 16 S rRNA types and 5 PFGE types found from all the strains.

CONCLUSIONSThree relative S. aureus strains were involved in the severe food-poisoning at least. Molecular subtyping might give a molecular epidemiological evidence and support the source tracing of an outbreak.

Bacterial Typing Techniques ; China ; Electrophoresis, Gel, Pulsed-Field ; Enterotoxins ; Humans ; Staphylococcal Food Poisoning ; epidemiology ; microbiology ; Staphylococcus aureus ; classification ; genetics ; isolation & purification

OBJECTIVETo investigate the contamination of Staphylococcus aureus isolates in ice cream by phenotypic typing and molecular typing.

METHODSThe Staphylococcus aureus isolates were separated from ice cream, filler, cutter, salves and material. The separated isolates were characterized by drug-resistance, staphylococcal enterotoxin (SEA-E), SE (A-E, G-J) genes and pulsed-field gel electrophoresis (PFGE) types.

RESULTTwo Staphylococcus aureus isolates were separated, one from ice cream, another from cutter. Their characteristics of drug-resistance, staphylococcal enterotoxin (SEA-E), SE (A-E,G-J) genes and PFGE type were the same.

CONCLUSIONThe two Staphylococcus aureus isolates were the same clone. The contaminated Staphylococcus aureus isolates could be traced to the contaminated cutters.

Anti-Bacterial Agents ; pharmacology ; Bacterial Typing Techniques ; Electrophoresis, Gel, Pulsed-Field ; Enterotoxins ; genetics ; Food Microbiology ; Ice Cream ; microbiology ; Microbial Sensitivity Tests ; Staphylococcus aureus ; classification ; drug effects ; isolation & purification

OBJECTIVETo investigate the homology of Staphylococcus aureus (SA) strains isolated from nose and skin lesions of impetigo children.

METHODSTotally 263 outpatients aged 3 months to 14 years who were seen by the Department of Dermatology of Beijing Children's Hospital between August 2005 and March 2006 were enrolled in this study. The isolations from nose and skin lesions of 58 impetigo children who were randomly selected from these 263 children with spa sequence were typed. The sequence results of SA were analyzed using special websites.

RESULTSThere were 106 impetigo patients in these 263 children. The isolation rate of SA was 78.3% in the nose of 106 impetigo patients and was 21.0% in that of the rest 157 patients (P < 0.01). The age of all nasal carriers was concentrated in 1-6 years. Among the 106 impetigo patients, 30 patients had their primary lesions on the face (including 28 cases of SA nose isolates) and 76 patients had their primary lesions on the other parts of body (including 56 cases of SA nose isolates) (P < 0.01). The spa typing showed that 26 of the 30 impetigo patients had the same type pairs of nose and skin.

CONCLUSIONSSA isolated from the skin lesions and nose of impetigo patients has remarkably homology. Nasal carriage of SA may be closely relevant with the occurrence of impetigo.

Adolescent ; Child ; Child, Preschool ; Female ; Humans ; Impetigo ; microbiology ; Infant ; Male ; Nasal Cavity ; microbiology ; Sequence Homology ; Skin ; microbiology ; Staphylococcus aureus ; classification ; genetics ; isolation & purification

OBJECTIVETo investigate the relation between the epidemiological strains of meticillin-resistant Staphylococcus aureus (MRSA) and the strains isolated from the nasal fossa of the medical staff and inpatients.

METHODSThe MRSA strains were isolated from the nasal fossa of the medical staff and inpatients in the Department of Neurosurgery. The genes of the isolated strains were amplified by randomly amplified polymorphic DNA (RAPD) assay.

RESULTSThree and 12 MRSA strains were isolated from the nasal fossa of the medical staff and patients who were hospitalized for more than 1 week, respectively, and RAPD assay revealed high homology between the isolated strains.

CONCLUSIONCross infection can be present between the medical staff, inpatients, and the infected patients.

China ; epidemiology ; Cross Infection ; microbiology ; DNA, Bacterial ; genetics ; isolation & purification ; Humans ; Infectious Disease Transmission, Professional-to-Patient ; Inpatients ; Medical Staff ; Methicillin Resistance ; Nasal Cavity ; microbiology ; Phylogeny ; Random Amplified Polymorphic DNA Technique ; Staphylococcal Infections ; epidemiology ; microbiology ; Staphylococcus aureus ; classification ; genetics ; isolation & purification

Panton-Valentine leukocidin (PVL)-positive USA300 clone has been the most successful community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) clone spreading in North America. In contrast, PVL-negative ST72-CA-MRSA has been predominant in Korea, and there has been no report of infections by the USA300 strain except only one case report of perianal infection. Here, we describe the first case of pneumonia caused by the USA300 strain following pandemic influenza A (H1N1) in Korea. A 50-year-old man was admitted with fever and cough and chest radiograph showed pneumonic consolidation at the right lower lung zone. He received a ventilator support because of respiratory failure. PCR for pandemic influenza A (H1N1) in nasopharyngeal swab was positive, and culture of sputum and endotracheal aspirate grew MRSA. Typing of the isolate revealed that it was PVL-positive, ST 8-MRSA-SCCmec type IV. The analysis of the PFGE patterns showed that this isolate was the same pulsotype as the USA300 strain.
Community-Acquired Infections/*etiology/microbiology ; Humans ; Influenza A Virus, H1N1 Subtype ; Influenza, Human/*complications ; Male ; *Methicillin-Resistant Staphylococcus aureus/classification/isolation & purification ; Middle Aged ; Pneumonia, Staphylococcal/*etiology/microbiology ; Republic of Korea ; Staphylococcal Infections/*etiology/microbiology

OBJECTIVETo investigate the molecular characteristics of methicillin-resistant Staphylococcus aureus (MRSA) isolates from Chinese children in seven cities.

METHODA total of 134 MRSA isolates were collected from nine hospitals. Multilocus sequence typing and spa typing were analyzed by polymerase chain reaction (PCR), and staphylococcal chromosomal cassette mec (SCCmec) type was analyzed by multiplex PCR. The Panton-Valentine leukocidin (pvl) gene was also detected.

RESULTMost MRSA strains were isolated from pneumonia and skin and soft tissue infection (SSTIs) patients, accounting for 82.1%. Overall, 16 sequence types (STs) were obtained, and CC59 (51.7%) was found to be the most prevalent, which included ST 59 and ST 338, followed by ST239 (16.4%). SCCmec types II, III, IV, and V were also identified in the current study. SCCmec type IV was the most predominant type at 50.0%, followed by SCCmec type V at 23.9% and III at 23.9%. SCCmec subtypes IVa, IVc, and IVg were found among SCCmec type IV strains, whereas IVa was the main subtype at 77.6%. Twenty-six spa types were also identified, among which the predominant type was t437 (47.8%). The prevalence of pvl genes and the SCCmec type of strain was relevant, and the pvl gene positive rate was higher in SCCmec type IV and V-type strains than in SCCmec type II and III strains (58.6% vs. 14.3%, P < 0.05); there was a significant difference between them. In the strains isolated from pneumonia and SSTIs, ST59-MRSA-IVa(t437) was the predominant clone. There were five clones detected from the strains isolated from septicemia, with ST59-MRSA-IVa(t437) and ST59-MRSA-V(t437) as the main clones (57.1%). Various predominant clones existed in different regions. ST59-MRSA-IVa(t437) was the prevalent clone in the Guangzhou, Beijing, Chongqing, and Shenzhen areas, whereas ST239-MRSA-III(t037) was the prevalent clone in the Shanghai area. Fifty percent of the isolates from the Wenzhou area belonged to ST910-MRSA-V(t318), whereas three clinical strains isolated from the Shenyang region belonged to three different types.

CONCLUSIONThe results indicate that MRSA isolates from Chinese children are largely associated with the ST59-MRSA-IV(t437) and ST239-MRSA-III(t037) clones. These two may belong to community-acquired MRSA and hospital-acquired ones, respectively. Different prevalent clones were detected in different diseases and different regions. Therefore, there is a need to conduct further research on clinical isolates, which can guide the choice of antibiotic treatment and the examination of MRSA prevalence.

Adolescent ; Bacterial Typing Techniques ; Child ; Child, Preschool ; China ; epidemiology ; DNA, Bacterial ; genetics ; Female ; Humans ; Infant ; Infant, Newborn ; Male ; Methicillin-Resistant Staphylococcus aureus ; classification ; genetics ; isolation & purification ; Prevalence ; Staphylococcal Infections ; epidemiology ; microbiology

OBJECTIVETo investigate the SCCmec genotyping, subtype and antimicrobial susceptibility tests in methicillin resistant staphylococcus aureus to guide the clinical treatment and provide the proof for molecular epidemiology.

METHODSTo detect mecA gene and SCCmec genetyping and subtype in 50 MRSA by PCR. According to CLSI's guideline, antimicrobial susceptibility tests were performed with disk diffusion.

RESULTSAll 50 MRSA had mecA genes. 45 strains were SCCmec III types; 3 strains were SCCmec III A types; 2 strains were SCCmec II types. There were no SCCmec I and SCCmec IV types. SCCmec II, SCCmec III and SCCmec III A type strains were all multiresistant.

CONCLUSION50 MRSA are all multiresistant. SCCmec III are the main types.

Anti-Bacterial Agents ; pharmacology ; Bacterial Proteins ; genetics ; China ; epidemiology ; Drug Resistance, Multiple, Bacterial ; Genotype ; Humans ; Methicillin-Resistant Staphylococcus aureus ; classification ; drug effects ; genetics ; isolation & purification ; Microbial Sensitivity Tests ; Penicillin-Binding Proteins ; Phylogeny ; Staphylococcal Infections ; epidemiology ; microbiology