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

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

OBJECTIVETo detect the enterotoxin genes of Staphylococcus aureus (SA) isolated from clinical specimens and analyze the correlation between enterotoxin genes and drug resistance of SA.

METHODSThe mecA gene and enterotoxin genes A-F of clinical SA isolates were identified by polymerase chain reaction (PCR), and the genes were sequenced to investigate the correlation of these genes to drug resistance.

RESULTSThe detection rate of enterotoxin genes was 100% in 67 methicillin- resistant SA (MRSA), showing no significant difference from the rate in 57 methicillin-sensitive SA (MSSA) (83.5%, χ(2)=0.203, P>0.05). Of the 116 strains carrying enterotoxin genes (93.5%), the detection rates of SEA, SEB, SEC, SED and SEF were 90.5%, 6.9%, 61.3%, 5.2%, 25.9% and 93.5%, respectively, and none of the strains were positive for SEE gene. In these strains, 78 (67.2%) carried 2 or more enterotoxin genes, and the main genotypes were SEA and SEC (33.6%), SEA and SEF (7.8%), and SEA and SEC and SEF (13.8%). Compared with the strains carrying a single enterotoxin gene, those with multiple enterotoxin genes showed a higher drug resistance rate, among which 75% of the SA strains carrying SEA+SEC+SEF were resistant to SXT, significantly higher than the rates of SA carrying SEA (28.6%) and SEA+SEC (38.7%) (P<0.05). The SA strains carrying SEA+SEC+SEF and SEA+SEF showed significantly higher amikacin resistance rates than SA strain carrying SEA (75.0%, 77.0%, 21.5%, respectively, P<0.05).

CONCLUSIONClinical isolates of SA carrying multiple enterotoxin genes have a higher drug resistance rate than those with a single enterotoxin gene, suggesting the the important role of enterotoxin in multidrug resistance.

Drug Resistance, Multiple, Bacterial ; genetics ; Enterotoxins ; genetics ; Humans ; Staphylococcal Infections ; microbiology ; Staphylococcus aureus ; drug effects ; genetics ; isolation & purification

The alpha-hemolysin protein of Staphylococcus aureus, which was expressed in Escherichia coli BL21 (DE3) with recombinant pET32a(+)-alpha-HL plasmid, was purified with gel filtration chromatography (GFC) and Ni-NTA spin columns. The quality and biological characteristic were compared. First, the purified products were analyzed with SDS-PAGE, and the expected protein band was with a molecular mass of 53 kD. Second, protein concentration was determined by the method of Bradford, and the median hemolytic dose potency (HD50) was finally analyzed with rabbit erythrocyte. The protein purified with GFC was 0.337 mg/mL, its hemolysis activity was 1519 HU/mg, and hemolysin yield was 14.04%. Meanwhile, the protein purified with the Ni-NTA Spin Columns was 0.35 mg/mL, its hemolysis activity was 1463 HU/mg, and hemolysin yield was 17.5%, respectively. The results showed that there is no significant difference in the quality, hemolysis activity and yield of the recombinant proteins purified with Ni-NTA spin columns and GFC.
Chromatography, Gel ; methods ; Escherichia coli ; genetics ; metabolism ; Hemolysin Proteins ; genetics ; isolation & purification ; Nitrilotriacetic Acid ; analogs & derivatives ; Organometallic Compounds ; Recombinant Proteins ; genetics ; isolation & purification ; Staphylococcus aureus ; genetics ; metabolism

OBJECTIVETo establish a systematic method for isolation and identification of aerobic and facultative anaerobic bacteria in the oral cavity.

METHODSSamples of the saliva, dental plaque and periapical granulation tissue were collected from 20 subjects with healthy oral condition and from 8 patients with different oral diseases. The bacteria in the samples were identified by morphological identification, VITEK automatic microorganism identification and 16s rRNA gene sequencing.

RESULTSVITEK automatic microorganism identification and 16s rRNA gene sequencing showed an agreement rate of 22.39% in identifying the bacteria in the samples. We identified altogether 63 bacterial genus (175 species), among which Streptococcus, Actinomyces and Staphylococcus were the most common bacterial genus, and Streptococcus anginosus, Actinomyces oris, Streptococcus mutans and Streptococcus mitis were the most common species. Streptococcus anginosus was commonly found in patients with chronic periapical periodontitis. Streptococcus intermedius and Staphylococcus aureus were common in patients with radiation caries, and in patients with rampant caries, Streptococcus mutans was found at considerably higher rate than other species.

CONCLUSIONAerobic and facultative anaerobic bacteria are commonly found in the oral cavity, and most of them are gram-positive. 16s rRNA gene sequencing is more accurate than VITEK automatic microorganism identification in identifying the bacteria.

Actinomyces ; isolation & purification ; Dental Caries ; Dental Plaque ; microbiology ; Humans ; Mouth ; microbiology ; RNA, Ribosomal, 16S ; genetics ; Saliva ; microbiology ; Staphylococcus aureus ; isolation & purification ; Streptococcus ; isolation & purification

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

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

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 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: We evaluated the performance of the BD MAX StaphSR Assay (SR assay; BD, USA) for direct detection of Staphylococcus aureus and methicillin resistance not only in S. aureus but also in coagulase-negative Staphylococci (CNS) from positive blood cultures. METHODS: From 228 blood culture bottles, 103 S. aureus [45 methicillin-resistant S. aureus (MRSA), 55 methicillin-susceptible S. aureus (MSSA), 3 mixed infections (1 MRSA+Enterococcus faecalis, 1 MSSA+MRCNS, 1 MSSA+MSCNS)], and 125 CNS (102 MRCNS, 23 MSCNS) were identified by Vitek 2. For further analysis, we obtained the cycle threshold (Ct) values from the BD MAX system software to determine an appropriate cutoff value. For discrepancy analysis, conventional mecA/mecC PCR and oxacillin minimum inhibitory concentrations (MICs) were determined. RESULTS: Compared to Vitek 2, the SR assay identified all 103 S. aureus isolates correctly but failed to detect methicillin resistance in three MRSA isolates. All 55 MSSA isolates were correctly identified by the SR assay. In the concordant cases, the highest Ct values for nuc, mecA, and mec right-extremity junction (MREJ) were 25.6, 22, and 22.2, respectively. Therefore, we selected Ct values from 0-27 as a range of positivity, and applying this cutoff, the sensitivity/specificity of the SR assay were 100%/100% for detecting S. aureus, and 97.9%/98.1% and 99.0%/95.8% for detecting methicillin resistance in S. aureus and CNS, respectively. CONCLUSIONS: We propose a Ct cutoff value for nuc/mec assay without considering MREJ because mixed cultures of MSSA and MRCNS were very rare (0.4%) in the positive blood cultures.
Anti-Bacterial Agents/pharmacology ; Bacteremia/diagnosis/microbiology ; Coagulase/metabolism ; Humans ; Methicillin-Resistant Staphylococcus aureus/drug effects/genetics/*isolation & purification ; Microbial Sensitivity Tests ; Oxacillin/pharmacology ; Reagent Kits, Diagnostic ; Staphylococcus/drug effects/enzymology/genetics/isolation & purification ; Staphylococcus aureus/drug effects/genetics/*isolation & purification