OBJECTIVEThis study aimed to establish the method of expression and purification of EsxB protein, explore the EsxB antibody-positive Staphylococcus aureus (S. aureus) clinical infection status and relevance of drug resistance.

METHODSConstructed EsxB prokaryotic expression system by homologous recombination, Ni(2+) column was used to purify EsxB protein; and then ELISA was used to detect the anti-EsxB antibodies in serum of 78 patients with S. aureus infection; antimicrobial susceptibility of related S. aureus strains by automatic bacterial identification analyzer.

RESULTSEsxB prokaryotic protein expression system was constructed and EsxB protein was purified successfully; anti-EsxB antibodies were present in the serum of patients with S. aureus infection up to 28.21% (22/78). The proportion of multi-drug resistant and Methicillin-resistant S. aureus strains isolated from anti-EsxB antibodies positive patients were 100.0% (22/22), 77.3% (17/22), respectively, which were statistically higher than those strains isolated from anti-EsxB antibody-negative patients (35.7% (20/56) and 21.4% (12/56), respectively) (all P values < 0.01).

CONCLUSIONMethod for expression and purification of EsxB protein was established. All the S. aureus strains isolated from EsxB antibody-positive patients were multidrug resistant strains and most of them were resistant to methicillin.

Anti-Bacterial Agents ; pharmacology ; Bacterial Proteins ; biosynthesis ; genetics ; isolation & purification ; Humans ; Methicillin ; pharmacology ; Methicillin-Resistant Staphylococcus aureus ; Microbial Sensitivity Tests ; Staphylococcus aureus ; isolation & purification

OBJECTIVETo investigate the genotypic diversity of Methicillin-resistant Staphylococcus aureus (MRSA) isolated from pigs and retail foods from different geographical areas in China and further to study the routes and rates of transmission of this pathogen from animals to food.

METHODSSeventy-one MRSA isolates were obtained from pigs and retail foods and then characterized by multi-locus sequencing typing (MLST), spa typing, multiple-locus variable number of tandem repeat analysis (MLVA), pulsed-field gel electrophoresis (PFGE), and antimicrobial susceptibility testing.

RESULTSAll isolated MRSA exhibited multi-drug resistance (MDR). Greater diversity was found in food-associated MRSA (7 STs, 8 spa types, and 10 MLVA patterns) compared to pig-associated MRSA (3 STs, 1 spa type, and 6 MLVA patterns). PFGE patterns were more diverse for pig-associated MRSA than those of food-associated isolates (40 vs. 11 pulse types). Among the pig-associated isolates, CC9-ST9-t899-MC2236 was the most prevalent clone (96.4%), and CC9-ST9-t437-MC621 (20.0%) was the predominant clone among the food-associated isolates. The CC9-ST9 isolates showed significantly higher antimicrobial resistance than other clones. Interestingly, CC398-ST398-t034 clone was identified from both pig- and food-associated isolates. Of note, some community- and hospital-associated MRSA strains (t030, t172, t1244, and t4549) were also identified as food-associated isolates.

CONCLUSIONCC9-ST9-t899-MC2236-MDR was the most predominant clone in pigs, but significant genetic diversity was observed in food-associated MRSA. Our results demonstrate the great need for improved surveillance of MRSA in livestock and food and effective prevention strategies to limit MDR-MRSA infections in China.

Animals ; Anti-Bacterial Agents ; pharmacology ; China ; Food Microbiology ; Humans ; Methicillin ; pharmacology ; Methicillin Resistance ; Methicillin-Resistant Staphylococcus aureus ; genetics ; isolation & purification ; Nose ; microbiology ; Swine ; microbiology

OBJECTIVETo genotypically characterize methicillin-resistant Staphylococcus aureus (MRSA) strains isolated from medical and surgical wards in Universiti Kebangsaan Malaysia Medical Centre (UKMMC) in 2009.

METHODSMRSA strains were collected and molecularly typed by pulsed-field gel electrophoresis (PFGE).

RESULTSPFGE typing on 180 MRSA isolated in UKMMC identified 5 pulsotypes (A-E) and 6 singletons, where pulsotypes B and C were suspected to be divergent clones originating from a single ancestor. This study also showed that most MRSA strains were isolated from swab (119 isolates), followed by blood (22 isolates), tracheal aspirate (11 isolates) and sputum (10 isolates). On the other hand, urine and bone isolates were less, which were 4 and 1 isolates, respectively. The distribution of different pulsotypes of MRSA among wards suggested that MRSA was communicated in surgical and medical wards in UKMMC, with pulsotype B MRSA as the dominant strain. Besides, it was found that most deceased patients were infected by pulsotype B MRSA, however, no particular pulsotype could be associated with patient age, underlying disease, or ward of admittance.

CONCLUSIONSFive pulsotypes of MRSA and 6 singletons were identified, with pulsotype B MRSA as the endemic strains circulating in these wards, which is useful in establishment of preventive measures against MRSA transmission.

Electrophoresis, Gel, Pulsed-Field ; Evolution, Molecular ; Hospitals ; Malaysia ; epidemiology ; Methicillin-Resistant Staphylococcus aureus ; genetics ; isolation & purification ; Staphylococcal Infections ; epidemiology ; microbiology

BACKGROUND: Susceptibility testing of Staphylococcus aureus often requires cumbersome supplementary tests. MicroScan Pos Breakpoint Combo Panel Type 28 (PBC28) (Siemens, USA) includes cefoxitin screening to detect methicillin-resistant Staphylococcus aureus (MRSA), inducible clindamycin resistance detection (ICD), and determination of low-range minimum inhibitory concentration of vancomycin (0.5-16 microgram/mL). The purpose of this study was to evaluate the performance of PBC28 in comparison with that of Pos Combo Type 1A (PC1A) (Siemens). METHODS: From December 2009 to March 2010, 500 non-duplicate clinical isolates of S. aureus were tested with PC1A and PBC28. Categorical agreements (CA) between the interpretations of the 2 panels were estimated. The presence of the mecA gene was determined by PCR, and double-disk diffusion test (D-test) was performed on the isolates resistant to erythromycin but susceptible or intermediately resistant to clindamycin. Ninety-six isolates representing various vancomycin minimum inhibitory concentrations (MICs) were tested in parallel with repeat PBC28, broth macrodilution, and epsilometer test (E test). RESULTS: The CA was 99.3% with a very major error (VME) of 0.2%, major error (ME) of 0.1%, and minor error (mE) of 0.4% in total. PBC28 showed 100% CA for 1 isolate with vancomycin MIC of 4 microgram/mL and 35 isolates (7.0%) with MIC of 2 microgram/mL. However, only 15, 27, and 35 isolates with vancomycin MIC of 2 microgram/mL showed 100% CA in repeat PBC28, broth macrodilution, and E test, respectively. PC1A and PBC28 detected all 314 mecA-positive isolates. Among the 63 isolates tested with the D-test, 58 (92.1%) were positive, and the results were 100% concordant with those of ICD. CONCLUSIONS: PBC28 can be appropriate susceptibility testing of S. aureus, including MRSA detection and ICD. However, the lower-range vancomycin MIC test was not reproducible enough to reliably differentiate MIC of 2 microgram/mL from MIC< or =1 microgram/mL.
Anti-Bacterial Agents/*pharmacology ; Bacterial Proteins/genetics ; Cefoxitin/*pharmacology ; Clindamycin/*pharmacology ; Drug Resistance, Bacterial ; Methicillin-Resistant Staphylococcus aureus/genetics/isolation & purification ; *Microbial Sensitivity Tests ; Reagent Kits, Diagnostic ; Sensitivity and Specificity ; Staphylococcus aureus/*drug effects/genetics/isolation & purification ; Vancomycin/*pharmacology

In this study, we investigated the genetic background of 70 Staphylococcus aureus isolates (36 methicillin-resistant S. aureus [MRSA] and 34 methicillin-susceptible S. aureus [MSSA]) obtained from blood at a Korean tertiary-care hospital, using spa typing, multilocus sequence typing, and SCCmec typing. In addition, the prevalence of enterotoxin (sea, seb, sec, sed, see, seg, seh, sei, and sek), tst, and pvl genes among the samples was assessed via polymerase chain reaction, and the results were compared with those of 95 isolates of S. aureus obtained from nasal swabs. All MRSA isolates from blood, except one, belonged to three major clones: sequence type (ST)5-MRSA-II, ST72-MRSA-II (or IVA), and ST239-MRSA-III, among which ST5-MRSA-II was the predominant clone. The prevalence of enterotoxin genes in the S. aureus isolates obtained from blood differed significantly from those from the nasal swabs for the sea, seb, sec, and seh gene. In particular, the seb and sec genes were detected exclusively in the MRSA isolates of ST5 or spa-CC002, thereby suggesting the co-adaptation of virulence genes with the genetic background and their contribution to biological fitness.
Bacteremia/*microbiology ; Enterotoxins/genetics ; Genotype ; Hospitals ; Humans ; Korea ; Methicillin-Resistant Staphylococcus aureus/genetics/*isolation & purification ; Microbial Sensitivity Tests ; Nose/*microbiology ; Staphylococcal Infections/diagnosis/*microbiology ; Staphylococcus aureus/genetics/*isolation & purification

Healthcare-associated infections caused by methicillin-resistant Staphylococcus aureus (MRSA) have recently become an important issue for healthcare facilities due to high rates of infection, mortality, and high treatment costs. We investigated the frequency of MRSA in healthcare workers (HCWs) via nasal carriage and assessed the performance of the LightCycler(R) MRSA Advanced test. We tested nasal swabs from the anterior nares of participating HCWs at an intensive care unit. Nasal swabs were identified as S. aureus, methicillin-sensitive or methicillin-resistant coagulase-negative staphylococci (MSCoNS or MRCoNS), or MRSA by using conventional culture and the LightCycler(R) MRSA Advanced test. Of the 142 HCWs who participated in this study, only 11 participants (7.8%) were MRSA-positive by conventional culture and MRSA ID, and 24 (16.9%) were positive for mecA by real time polymerase chain reaction (PCR). In terms of diagnostic performance, the LightCycler(R) MRSA Advanced test had a sensitivity of 100%, a specificity of 90.1%, a positive predictive value of 45.8%, and a negative predictive value of 100% compared with conventional culture method. The detection limit of the LightCycler(R) MRSA Advanced test was 103 colony/mL. We concluded that real-time PCR was able to rapidly and sensitively detect MRSA in HCWs. However, MRSA must be confirmed by culture due to false positivity.
Bacterial Proteins/genetics ; False Positive Reactions ; Humans ; Methicillin-Resistant Staphylococcus aureus/genetics/*isolation & purification ; Nurses ; Physicians ; Real-Time Polymerase Chain Reaction/methods ; Sensitivity and Specificity ; Staphylococcal Infections/*diagnosis

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

OBJECTIVETo study the relationship between nasal carriage and Staphylococcus aureus (S. aureus) infection in hospitalized children.

METHODSFifty-six hospitalized children infected with S. aureus were recruited in this study. Nasal swabs were collected and cultured, and the nasal carriage rate of S. aureus was examined. PVL virulence gene and mecA resistance gene were both detected in clinical strains and nasal carriage strains by PCR.

RESULTSTwenty-two (39%) of the 56 children had nasal carriage of S. aureus, and most of them (18 cases) were younger than one year. Among these 22 children, 11 (50%) had previous hospitalization over the past year. In the infected strains, the rate of methicillin-resistant S. aureus (MRSA) was 29% (16/56), while it was 32% (7/22) in carriage strains. The mecA positive results in clinical strains were consistent with the results in nasal carriage strains. Among 5 PVL-positive nasal carriage strains, 4 (90%) could be matched with their clinical strains, all of which were MRSA.

CONCLUSIONSNasal carriage is a potential risk factor for S. aureus infection. Nosocomial transmission may lead to nasal carriage, which can cause S. aureus infection. The isolation rate of MRSA is high in hospitalized children infected with S. aureus, which implies that more attention is needed for this situation. The isolates from noses may be clonally identical to the isolates from clinical secretions, and the homology between them needs to be confirmed by multi-locus sequence typing.

Bacterial Proteins ; genetics ; Carrier State ; microbiology ; Child ; Child, Hospitalized ; Child, Preschool ; Female ; Humans ; Infant ; Infant, Newborn ; Male ; Methicillin-Resistant Staphylococcus aureus ; isolation & purification ; Nose ; microbiology ; Penicillin-Binding Proteins ; Staphylococcal Infections ; microbiology ; Staphylococcus aureus ; isolation & purification

Community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) is an increasingly common worldwide and colonizing S. aureus strains may serve as the causative pathogen for overt clinical infections. This study was performed to determine whether the pathogenic CA-MRSA isolate in clinical infections was genetically related to the MRSA isolates in community carriers. We prospectively collected a total of 42 CA-MRSA isolates (23 clinical infection isolates and 19 colonization isolates) in a local region of Korea. Antimicrobial susceptibility tests, staphylococcal toxin assays, SCCmec typing, multilocus sequence typing (MLST), and spa (staphylococcal protein A) typing were performed with all isolates. Thirty-four (81%) of 42 CA-MRSA isolates belonged to sequence type (ST) 72 in the MLST analysis. The distribution of STs did not differ significantly between colonization and clinical infection isolates (89.5% [17/19] vs. 73.9% [17/23], P=0.26). Among the ST72-MRSA isolates, spa type t664 (18, 52.9%) and t324 (8, 23.5%) were common in both groups. This study demonstrates that the community-associated MRSA strains from patients with clinical infections are closely related to the strains found in carriers from one local community.
Community-Acquired Infections/microbiology ; Genotype ; Humans ; Methicillin-Resistant Staphylococcus aureus/*genetics/isolation & purification ; Microbial Sensitivity Tests ; Prospective Studies ; Republic of Korea ; Staphylococcal Infections/*microbiology

BACKGROUND: The BD GeneOhm MRSA PCR assay (Becton Dickinson, USA) is a qualitative real-time PCR test for rapid detection of nasal colonization of methicillin-resistant Staphylococcus aureus (MRSA). We evaluated the performance of BD GeneOhm MRSA PCR assay versus MRSASelect (Bio-Rad, France) and broth enrichment cultures for detection of MRSA from nasal swabs. METHODS: From August 2008 to January 2009, 295 nasal swabs were taken from patients in intensive care units and transported to the laboratory with BD CultureSwab Liquid Stuart Single Swab (Becton Dickinson, USA). The swabs were inoculated onto MRSASelect first and then suspended into GeneOhm sample buffer: 100 microliter of the suspension was inoculated into 6.5% NaCl-tryptic soy broth (Becton Dickinson, USA), which was subcultured on MRSASelect after overnight incubation (TSBS). Performances of GeneOhm MRSA and MRSASelect were compared to TSBS. RESULTS: With GeneOhm MRSA, 125 swabs (44.6%) were positive for MRSA, 13 (4.4%) were unresolved, and 2 were not determined. With MRSASelect and TSBS 86 (29.4%) and 106 swabs (36.2%), respectively, were positive. The sensitivity, specificity, and positive and negative predictive value of GeneOhm MRSA were 85.8%, 77.5%, and 72.8% and 93.5%, respectively, and corresponding values for MRSASelect were 78.3%, 94.8%, and 96.5% and 88.9%. Of the 33 patients whose 34 specimens were found false positive in GeneOhm MRSA, 23 patients were MRSA-positive either previously or subsequently to this study. All of the 10 patients with false-negative specimens in GeneOhm MRSA PCR assay were previously MRSA or methicilln-resistant coagulase negative staphylococci (MRCNS)-positive and were treated for MRSA, but they became MRSA-positive after 1 to 4 negative surveillance cultures. CONCLUSIONS: GeneOhm MRSA PCR assay showed a relatively high negative predictive value. However, its low specificity and frequent occurrence of unresolved results would be problematic in the endemic areas with a high prevalence of MRSA.
Endemic Diseases ; Humans ; Intensive Care Units ; Methicillin-Resistant Staphylococcus aureus/genetics/*isolation & purification ; Nose/*microbiology ; Polymerase Chain Reaction/*methods ; Reagent Kits, Diagnostic ; Sensitivity and Specificity ; Staphylococcal Infections/*diagnosis/epidemiology