We established a rapid detection method of New Delhi Metallo-beta-Lactamase Gene (NDM-1) based on Loop-mediated Isothermal Amplification (LAMP). With the application of LAMP, we designed four sets of LAMP premiers, using NDM-1 gene as the target sequence, and selected the set of optimal primers. Meanwhile, we established optimal reaction systems and conditions to carry out the sensitivity and specificity experiments. The experiment results showed that the whole detection process took only one hour and could be observed visually. In the experiment of sensitivity, NDM-1 gene had a detection limit of 6 copies in each reaction. In the experiment of specificity, we detected NDM-1 gene in 4 pathogen strains (Klebsiella pneumoniae, Escherichia coli, Staphylococcus aureus, Streptococcus pneumoniae), and the total DNA from intestinal microbes and the total DNA from soil microbes. We had not detected the amplification reactions. The detection method established could rapidly detect NDM-1 gene and visualize the experiment result. The method is easy to operate and has high sensitivity and specificity and thus has great application value in basic research laboratories, emergent detection and spot detection.
Bacteria ; enzymology ; genetics ; Bacteriological Techniques ; methods ; DNA, Bacterial ; genetics ; Escherichia coli ; enzymology ; genetics ; Klebsiella pneumoniae ; enzymology ; genetics ; Nucleic Acid Amplification Techniques ; methods ; Sensitivity and Specificity ; Staphylococcus aureus ; enzymology ; genetics ; Streptococcus pneumoniae ; enzymology ; genetics ; beta-Lactamases ; genetics

Lysostaphin, a specific endopeptidase enzyme derived from Staphylococcus aureus, is a bactericidal agent against Staphylococcus and difficult to be drug-resistant. This study established the monoclonal antibody affinity chromatography to obtain lysostaphin of high purity for drug-use standard. The purified Lysostaphin was of > 95% purity and its recovery rate more than 90%. Moreover, the affinity column kept its efficiency of purification invariable after more than 30 times repeat. Also, the dye release assay validated that the purified lysostaphin had significant bactericidal activity. This method was simple and of high efficacy for the lysostaphin purification and showed its potency in commercial production.
Antibodies, Monoclonal ; immunology ; Chromatography, Affinity ; methods ; Lysostaphin ; biosynthesis ; isolation & purification ; Recombinant Proteins ; biosynthesis ; isolation & purification ; Staphylococcus aureus ; enzymology

OBJECTIVETo evaluate the variety of non-myeloperoxidase-mediated system activity of neutrophils in newborns during bacterial infection and the effect of cord plasma on the activation of non-myeloperoxidase-mediated system.

METHODSAn infection model with Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) and a non-infection model with phorbol-12-myristate-13-acetate (PMA) were established to investigate the activation of non-myeloperoxidase-mediated system in neutrophils. According to the intensity of fluorescence, the activation of non-myeloperoxidase-mediated system of neutrophils was detected by flow cytometry (FCM). The blood cells and plasma were separated from cord blood and adult blood and cross-mixed in order to investigate the opsonic activity.

RESULTSIn the non-infection model, the activation of non-myeloperoxidase-mediated system with PMA stimulation in cord blood was lower compared with that in adult blood, the statistical difference was significant (t = 3.378, P < 0.01). In the infection model, the activations of non-myeloperoxidase-mediated system in cord blood were also lower compared with those in adult blood, while the statistical difference could only be found in the model with E. coli stimulation (t = 12.150, P < 0.001). Furthermore the experiments demonstrated that cord plasma could deeply depress the non-myeloperoxidase-mediated system activity with E. coli stimulation. On the contrary, adult plasma could successfully recruit the potential of non-myeloperoxidase-mediated system activity of neutrophils in newborns.

CONCLUSIONThe function of neonatal neutrophils might not developed very well. As a stimulant, E. coli failed to induce the non-myeloperoxidase-mediated system activity in neonates, which might be related to the lower level of immunoglobulins in cord blood. This result indicated that immunoglobulins played a more important modulating role in bacterial killing during gram-negative bacterial infections.

Escherichia coli ; immunology ; Fetal Blood ; immunology ; Flow Cytometry ; Humans ; Infant, Newborn ; Neutrophils ; enzymology ; immunology ; Peroxidase ; metabolism ; Staphylococcus aureus ; immunology

The emergence of multi-drug resistant gram-positive cocci such as methicillin-resistant (MR) staphylococci, vancomycin-resistant (VR) enterococci, and vancomycin-intermediate resistant S. aureus (VISA) has given new urgency to the development of new antimicrobial agents. One of these is quinupristin/dalfopristin (Q/D). We decided to determine the susceptibility of gram-positive cocci isolated at two university hospitals in Seoul to Q/D and compare the results with eight other antimicrobial agents. We investigated 120 isolates of S. aureus including 49 MRSAs and one VISA, 120 isolates of coagulase negative staphylococci (CNS), 64 E. faecalis and 56 E. faecium, including seven strains of VR E. faecium. Minimum inhibitory concentrations (MICs) and minimal bactericidal concentrations (MBCs) for several antimicrobials, including vancomycin and Q/D, were determined by broth microdilution. All S. aureus including VISA were susceptible to Q/D. Q/D MIC90 for both methicillin-susceptible S. aureus (MSSA) and MRSA was 0.25 g/mL. 49 (87.5%) of 56 E. faecium including six of seven VR E. faecium were susceptible to Q/D. E. faecalis were not susceptible to Q/D (only 1.5% susceptible), but were inhibited by ampicillin (94% susceptible) or vancomycin (95%). CNS was susceptible to Q/D (96% susceptible) and vancomycin (100% susceptible). One of 38 staphylococci and two of 17 E. faecium were tolerant to Q/D. In conclusion, Q/D showed excellent activity against all species of gram-positive cocci including MRSA, VISA, and VR E. faecium except E. faecalis, and may provide a valuable option for the treatment of infections caused by these emerging nosocomial pathogens of gram-positive cocci.
Antibiotics/pharmacology* ; Antibiotics, Peptide/pharmacology* ; Coagulase/analysis ; Enterococcus faecalis/drug effects ; Enterococcus faecium/drug effects ; Human ; Korea ; Microbial Sensitivity Tests* ; Staphylococcus/enzymology ; Staphylococcus/drug effects ; Staphylococcus aureus/drug effects ; Support, Non-U.S. Gov'tn ; Virginiamycin/pharmacology* ; Virginiamycin/analogs & derivatives*

Necrotizing fasciitis (NF) is a deep infection of the subcutaneous tissue that progressively destroys fascia and fat; it is associated with systemic toxicity, a fulminant course, and high mortality. NF most frequently develops from trauma that compromises skin integrity, and is more common in patients with predisposing medical conditions such as diabetes mellitus, atherosclerosis, alcoholism, renal disease, liver disease, immunosuppression, malignancy, or corticosteroid use. Most often, NF is caused by polymicrobial pathogens including aerobic and anaerobic bacteria. NF caused by Staphylococcus aureus as a single pathogen, however, is rare. Here we report a case of NF that developed in a healthy woman after an isolated shoulder sprain that occurred without breaking a skin barrier, and was caused by Staphylococcus aureus as a single pathogen.
*Arm ; Coagulase/metabolism ; Fasciitis, Necrotizing/*etiology/microbiology/pathology/surgery ; Female ; Humans ; Middle Aged ; Shoulder Joint/*injuries ; Sprains and Strains/*complications ; Staphylococcal Infections/*etiology/microbiology ; Staphylococcus aureus/enzymology/isolation & purification

Amino Acid Sequence ; Base Sequence ; Genes, Bacterial ; Humans ; Molecular Sequence Data ; Polymerase Chain Reaction ; Staphylococcus epidermidis ; enzymology ; genetics ; beta-Lactamases ; chemistry ; genetics

A N-acetylneuraminate lyase gene (shnal) from Staphylococcus hominis was cloned into pET-28a and expressed in Escherichia coli BL21 (DE3) host cells. The recombinant enzyme was purified and characterized. It is a homotetrameric enzyme with the optimum pH at 8.0 for the cleavage direction and the optimum pH and temperature were 7.5 and 45 degrees C for the synthetic direction. The activity of ShNAL is stable when incubated at 45 degrees C for 2 h but decreased rapidly over 50 degrees C. ShNAL showed high stability in a wide range pH from 5.0 to 10.0 with the residual activity being > 70% when the enzyme was incubated in different buffers at 4 degrees C for 24 h. Its K(m) towards N-acetylneuraminic acid, pyruvate and ManNAc were (4.0 +/- 0.2) mmol/L, (35.1 +/- 3.2) mmol/L and (131.7 +/- 12.1) mmol/L, respectively. The k(cat)/K(m) value of Neu5Ac, ManNAc, and Pyr for ShNAL were 1.9 L/(mmol x s), 0.08 L/(mmol x s) and 0.08 L/(mmol x s), respectively.
Bacterial Proteins ; genetics ; metabolism ; Cloning, Molecular ; Enzyme Stability ; Escherichia coli ; genetics ; metabolism ; Hydrogen-Ion Concentration ; Oxo-Acid-Lyases ; genetics ; metabolism ; Recombinant Proteins ; genetics ; metabolism ; Staphylococcus hominis ; enzymology ; Temperature

Peptide cyclization, a pivotal approach to modifying linear precursors of proteins and pepticles, has been used to enhance their biological activities and serum stabilities. Recently, sortase A (SrtA) from Staphyloccus aureus becomes a promising new technology for efficiently incorporating site specific modifications into proteins, conjugating the cell surface and cyclizing the linear peptides. In this study, we constructed two recombinant expression systems, one with chitin binding domain and the other with six-histidine tag and chitin binding domain on the N-terminal of SrtA, separately. The results of enzymatic kinetics indicate that the two recombinant tags do not impair the transpeptidase activity of SrtA compared with the standard reaction reported under the same reaction condition. The two synthesized peptides with N-ternimal three glycines and C-terminal penta-amino acid motif, LPETG, were cyclized using immobilized and recycled SrtA. The SrtA-based cyclization promises to represent a simple method for easy and efficient enzymatic synthesis of large cyclic peptides.
Aminoacyltransferases ; metabolism ; Bacterial Proteins ; metabolism ; Cyclization ; Cysteine Endopeptidases ; metabolism ; Enzymes, Immobilized ; metabolism ; Kinetics ; Peptides ; metabolism ; Peptides, Cyclic ; biosynthesis ; Staphylococcus aureus ; enzymology

BACKGROUNDGram-positive bacteria such as Staphylococcus aureus have been a common cause of infection among liver transplant (LT) recipients in recent decades. The understanding of local epidemiology and its evolving trends with regard to pathogenic spectra and antibiotic susceptibility is beneficial to prophylactic and empiric treatment for LT recipients. This study aimed to investigate etiology, timing, antibiotic susceptibility and risk factors for multidrug resistant (MDR) Gram-positive coccal bacteremia after LT.

METHODSA cohort analysis of prospectively recorded data was performed to investigate etiologies, timing, antibiotic susceptibility and risk factors for MDR Gram-positive coccal bacteremia in 475 LT recipients.

RESULTSIn 475 LT recipients in the first six months after LT, there were a total of 98 episodes of bacteremia caused by Gram-positive cocci in 82 (17%) patients. Seventy-five (77%) bacteremic episodes occurred in the first post-LT month. The most frequent Gram-positive cocci were methicillin-resistant coagulase-negative staphylococcus (CoNS, 46 isolates), methicillin-resistant Staphylococcus aureus (MRSA, 13) and enterococcus (34, E. faecium 30, E. faecalis 4). In all Gram-positive bacteremic isolates, 59 of 98 (60%) were MDR. Gram-positive coccal bacteremia and MDR Gram-positive coccal bacteremia predominantly occurred in patients with acute severe exacerbation of chronic hepatitis B and with fulminant/subfulminant hepatitis. Four independent risk factors for development of bacteremia caused by MDR Gram-positive coccus were: LT candidates with encephalopathy grades II - IV (P = 0.013, OR: 16.253, 95%CI: 1.822 - 144.995), pre-LT use of empirical antibiotics (P = 0.018, OR: 1.029, 95%CI: 1.002 - 1.057), post-LT urinary tract infections (P < 0.001, OR: 20.340, 95%CI: 4.135 - 100.048) and abdominal infection (P = 0.004, OR: 2.820, 95%CI: 1.122 - 10.114). The main infectious manifestations were coinfections due to gram-positive cocci and gram-negative bacilli.

CONCLUSIONSMethicillin-resistant CoNS and enterococci are predominant pathogens among LT recipients with Gram-positive coccal bacteremia. Occurrences of Gram-positive coccal bacteremia may be associated with the severity of illness in the perioperative stage.

Anti-Bacterial Agents ; pharmacology ; Bacteremia ; etiology ; microbiology ; Coagulase ; metabolism ; Drug Resistance, Multiple, Bacterial ; Enterococcus ; drug effects ; enzymology ; physiology ; Gram-Positive Bacterial Infections ; enzymology ; microbiology ; transmission ; Humans ; Liver Diseases ; microbiology ; Liver Transplantation ; adverse effects ; Risk Factors ; Staphylococcal Infections ; enzymology ; microbiology ; transmission ; Staphylococcus ; drug effects ; enzymology ; physiology

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