Six 89bp primers were designed on the base of the cDNA sequence encoding the human leptin reported on the NCBI. The synthetic gene with 464bp encoding rhLep was obtained by SOE ( splicing by overlap extension) PCR. The expression vector pET22b(+)/rhLep was constructed and transformed into E. coli BL21 (DE3). The rhLep protein was expressed as inclusion bodies with the yield of more than 50% of total bacterial proteins after IPTG induction. The rhLep protein, which has a molecular weight about 16kD, was purified by Ni2+ affinity chromatography column and identified by SDS-PAGE. The MTT Assay shows that rhLep promotes EC304 cells growth at the low concentration of 10ng/mL to 30 ng/mL, and rhLep appears cytotoxic to EC304 cells with the high dose of 50ng/mL to 225ng/mL. The viability of EC304 cells decreases to 1.2% with the concentration of 225ng/mL of rhLep. The massive apoptosis of rhLep on EC304 cells is observed by AO-staining under fluorescent microscope. All these results would lay the foundation for the further study of its biological functions in vitro and in vivo.
Apoptosis ; drug effects ; Escherichia coli ; genetics ; Humans ; Leptin ; genetics ; pharmacology ; Recombinant Proteins ; biosynthesis ; isolation & purification ; pharmacology

Lysins are murein hydrolases produced by bacteriophage that act on the cell wall of host bacteria to release progeny phages. Research indicated that lysins could kill bacteria effectively and specifically in vitro. To prepare recombinant bacteriophage lysin of Streptococcus (PlyC) and analyze its biological activity, we obtained two genes of PlyC named PlyCA and PlyCB by PCR amplification and inserted them into pET-32a(+), then transformed the recombinant expression vectors pET-32a(+)-PlyCA and pET-32a(+)-PlyCB into E. coli BL21(DE3) respectively. After induction with 0.7 mmol/L IPTG at 30 degrees C for 7 h, PlyCA and PlyCB were successfully expressed, SDS-PAGE analysis determined that they all constituted above 30% of the total cell proteins. After Ni(2+)-NTA affinity chromatography, the purity was more than 95%. With the denaturation and protein refolding, we gained the recombinant PlyC. To determine its biological activity, we adopted turbidimetry and plate count method. Before and after lysin treatment, the cell morphology was studied by scanning electron microscopy (SEM). The results showed that the recombinant PlyC could specifically cleavage Streptococcus pyogenes (group A beta-hemolytic streptococci). Under the incubation time of 60 min with 4 microg/mL PlyC in Streptococcus pyogenes dilution which OD600 was 0.56, the germicidal effect was up to 99.6%, while SEM observations showed that cell wall cracked and presented cell debris. This finding laid the foundation for the further study and achieving an effective treatment for streptococcal infection.
Bacteriolysis ; Enzymes ; biosynthesis ; genetics ; isolation & purification ; Escherichia coli ; genetics ; metabolism ; Recombinant Proteins ; biosynthesis ; genetics ; isolation & purification ; Streptococcus pyogenes ; drug effects

OBJECTIVETo study the genotype distribution of extended-spectrum beta-lactamases (ESBLs) in ESBLs-producing Escherichia coli (E. coli) isolates from posthepatitic cirrhosis' patients with bloodstream infection.

METHODSE. coli were isolated in bloodstream from patients with posthepatitic cirrhosis between January and December in 2011. The strains were identified by VITEK-II. The antibiol susceptibility tests were performed with K-B method. beta-lactamases genes were detected multi-PCR, PCR, sequence and blast.

RESULTSA total of 79 non-duplicate clinical isolates of E coli were consecutively collected from liver cirrhosis' patients with bloodstream infection. There were 20 isolates produced TEM-1 type beta-lactamases and 1 isolate produced SHV-1 typebeta-lactamases. 40 clinical isolates were detected to produce CTX-M type ESBLs, there were 20 CTX-M-1 group and 26 CTX-M-9 group, including 6 stains habouring both CTX-M-1 and CTX-M-9 group. Eight CTX-M genotypes were confirmed by sequencing of the PCR products, including CTX-M-3, CTX-M-14, CTX-M-15, CTX-M-24, CTX-M-28, CTX-M-31, CTX-M-65 and CTX-M-79.

CONCLUSIONCTX-M genotype ESBLs was the most popular extended-spectrum beta-lactamases in E. coli isolated from liver cirrhosis' patients with bloodstream infection. The CTX-M-14 is the dominant epidemic type.

Bacteremia ; microbiology ; Cross Infection ; microbiology ; Drug Resistance, Bacterial ; Escherichia coli ; drug effects ; enzymology ; genetics ; isolation & purification ; Escherichia coli Infections ; microbiology ; Escherichia coli Proteins ; genetics ; Genotype ; Hospitalization ; statistics & numerical data ; Humans ; Liver Cirrhosis ; therapy ; Microbial Sensitivity Tests ; beta-Lactamases ; genetics ; metabolism

OBJECTIVETo investigate the possible differences in antimicrobial resistance of Escherichia coli isolated from different samples in children.

METHODSSix hundred and twenty-nine samples from urine, sputum, blood and secretion were collected from June 2004 to May 2009 for bacterial identification by VITEK-32 automatic system and antimicrobial susceptibility tests by Kirby-Bauer method. The drug resistance rate of Escherichia coli isolated from different samples was compared.

RESULTSTwo hundred and sixty strains of Escherichia coli were isolated , and 108 of which were from urine , 64 from sputum, 54 from secretion and 23 from blood. ESBLs were detected in 96 (36.9%) of the 260 isolates, AmpC enzymes in 32 (12.3%), and ESBLs+AmpC in 8 (3.1%). The ESBLs positive rate of Escherichia coli isolates from sputum was significantly higher than that from other samples (P<0.05). The antimicrobial resistance rate of Escherichia coli strains from different samples to amoxicillin/clavulanic acid, ticarcillin/clavulanic acid, piperacillin, cefotaxime, cefuroxime, cefepime, gentamicin, cotrimoxazole, and nitrofurantoin was different. The resistance rate of the strains from sputum samples was higher than that from the other samples (P<0.05).

CONCLUSIONSEscherichia coli isolated from different samples have different antimicrobial resistance rates in children, so the selection of antibiotics for infections confirmed by bacterial cultures from different samples should based on drug sensitivity results.

Adolescent ; Child ; Child, Preschool ; Drug Resistance, Bacterial ; Escherichia coli ; drug effects ; enzymology ; isolation & purification ; Female ; Humans ; Male ; beta-Lactamases ; analysis

INTRODUCTIONAt the time of the study, 3 plasmid-borne qnr determinants (qnrA, qnrB and qnrS) and 1 plasmid-borne aminoglycoside-modifying enzyme determinant that confers quinolone resistance (aac(6')-Ib-cr) had been described in the literature.

MATERIALS AND METHODSWe studied the prevalence of the 3 qnr determinants in a total of 117 nalidixic acid-resistant urinary isolates of Klebsiella pneumoniae (61 isolates) and Escherichia coli (56 isolates) using multiplex polymerase chain reaction (PCR). Further, a subset of the original strains (comprising 14 E. coli and 38 K. pneumoniae) showing reduced susceptibility to the aminoglycosides underwent PCR for aac(6')-Ib, followed by restriction digestion with BtsCI to detect the variant aac(6')-Ib-cr.

RESULTSTwenty-eight of 61 (45.9%) Klebsiella isolates were found to possess at least 1 qnr determinant. Only 1/56 (1.8%) E. coli isolates were found to possess a qnr determinant. Two of the Klebsiella isolates possessed 2 qnr determinants each (qnrB and qnrS). The predominant determinant was qnrB (19 isolates). There were 11 isolates harbouring qnrS, and only 1 with qnrA. 1/14 (7.1%) E. coli and 35/38 K. pneumoniae (92.1%) were found to possess aac(6')-Ib-cr. There was pairwise association between each of qnr, aac(6')-Ib-cr and the presence of an extended-spectrum beta-lactamase.

CONCLUSIONSA high prevalence of plasmid-mediated quinolone resistance determinants [i.e., qnrS, qnrB and aac(6')-Ib-cr] was found in quinolone-resistant K. pneumoniae isolated in a large hospital in Singapore.

Drug Resistance, Bacterial ; Escherichia coli ; drug effects ; isolation & purification ; Hospitals ; Humans ; Klebsiella pneumoniae ; drug effects ; isolation & purification ; Molecular Sequence Data ; Quinolones ; pharmacology ; R Factors ; Singapore ; Urine ; microbiology

OBJECTIVETo investigate the distribution of burn pathogens and their antibiotic resistance in a burn unit, so as to provide reference for clinical practice.

METHODSThree hundred and forty-eight burn patients hospitalized in our department were enrolled in this study. The pathogens isolated from the wounds, blood, venous catheter, sputum, urine, purulent discharge of wounds in these patients, and their antibiotic resistance were surveyed by retrospective analysis from Jan, 2001 to Dec, 2006.

RESULTSTotal-ly 464 strains were isolated, among which Gram negative (G-) bacilli accounted for 52.6%, Gram positive microorganisms (G+) accounted for 40.5%, and fungi accounted for 6.9%. The main pathogens were Staphylococcus aureus, Pseudomonas aeruginosa, Acinetobacter species and Escherichia coli, among which Staphylococcus aureus (MRSA) was predominant (93.5%). MRSA was 100% resistant to levofloxacin, penicillium, oxacillin, and it was also resistant to other antibiotics except Vancomycin. The resistance rate of Pseudomonas aeruginosa to Cefoperazone/Sulbactam, Imipenem and cefepime were 15.8%, 36.8%, 33.3%, respectively.

CONCLUSIONStaphylococcus aureus, Pseudomonas aeruginosa, Acinetobacter species and Escherichia coli were predominant in the burn unit,among them Staphylococcus aureus and Acinetobacter were more resistant to antibiotics.

Acinetobacter baumannii ; drug effects ; isolation & purification ; Burn Units ; Burns ; microbiology ; Cross Infection ; microbiology ; Drug Resistance, Bacterial ; Escherichia coli ; drug effects ; isolation & purification ; Humans ; Pseudomonas aeruginosa ; drug effects ; isolation & purification ; Retrospective Studies ; Staphylococcus aureus ; drug effects ; isolation & purification

OBJECTIVETo screen effective constituents of Discocleidion rufescens against bacteria.

METHODCompounds were isolated by bioassay-guided fractionation method with various chromatographic methods and their structures were determined by spectral analysis and chemical evidence.

RESULTSeven compounds were isolated from the Et2O and n-BuOH extracts and identified as 5,7-dihydroxy-4'-methoxy flavone (1), quercetin (2), apigenin-7-O-beta-D-glucopyranoside (3), apigenin-7-O-neohesperidoside (4), luteolin-7-O-neohesperidoside (5), gallic acid (6) and beta-daucosteol (7).

CONCLUSIONCompounds 1-6 were isolated from genus Discocleidion for the first time and exhibited certain inhibitory activity against Escherichia coli. Compound 6 also showed antibacterial activity against Staphylococus aureus.

Anti-Bacterial Agents ; analysis ; isolation & purification ; pharmacology ; Escherichia coli ; drug effects ; Euphorbiaceae ; chemistry ; Plant Extracts ; analysis ; isolation & purification ; pharmacology ; Staphylococcus aureus ; drug effects

Bacteria ; drug effects ; isolation & purification ; Drug Resistance ; Escherichia coli ; drug effects ; Female ; Haemophilus influenzae ; drug effects ; Humans ; Klebsiella pneumoniae ; drug effects ; Male ; Microbial Sensitivity Tests ; Respiratory Tract Infections ; microbiology ; Staphylococcus aureus ; drug effects ; Streptococcus pneumoniae ; drug effects

OBJECTIVETo analyze the distribution and drug resistance of bacteria in different wound infections and provide evidence for wound infection control in subtropical regions.

METHODSThis study involved 265 patients from 4 different departments of our hospital who experienced wound infections between July, 2007 and July, 2008. The bacterial strain distribution in the wounds and drug resistance of the bacteria were analyzed.

RESULTSAcinetobacter baumanii (39% of the total strain identified) was the most frequent bacterial strain causing infection of the burn wounds, followed by Proteus mirabilis (20%) and Pseudomonas aeruginosa (20%). E. coli infection was prevalent in the departments of general surgery (37%) and urinary surgery (64%), and Pseudomonas aeruginosa and Pseudomonas pneumonia infections were detected at the rate of 30% and 43% in the urinary surgery department, respectively. Different bacterial strains were found at similar rates around 10% in the wounds of patients undergoing traumatic surgery.

CONCLUSIONDespite that the commonly seen pathogenic bacteria in burn patients including Staphylococcus aureus have been effectively controlled by early application of antibiotics, the opportunistic pathogens such as Acinetobacter baumanii and Proteus mirabilis often survive these antibiotics, and some strains evolve to be drug-resistant and even multi-drug-resistant. E. coli infection is prevalent in general surgery and urinary surgery departments, where Staphylococcus aureus and Pseudomonas aeruginosa infections can also be found frequently. All kinds of bacteria infection are present in trauma surgery department, each found at the rate around 10%.

Acinetobacter baumannii ; drug effects ; isolation & purification ; Adolescent ; Adult ; Burns ; complications ; Child ; Child, Preschool ; Drug Resistance, Bacterial ; Escherichia coli ; drug effects ; isolation & purification ; Female ; Humans ; Male ; Middle Aged ; Pseudomonas aeruginosa ; drug effects ; isolation & purification ; Wound Infection ; etiology ; microbiology ; Young Adult

Marine Streptomyces GB-2, isolated from marine samples collected in the intel tidal zone of Lianyungang, was found to produce antibacterial substance which exhibited significant inhibitory effects on 11 Gram-positive bacteria and 4 Gram-negative bacteria. The antibacterial substance was proved to be neutral and water-soluble according to paper chromatogram analysis, and its production was significantly associated with aritificial seawater. The stability analysis of the fermentation broth of Streptomyces GB-2 showed that it was very stable at pH1 and pH12 under 121 degrees C and changed very little under ultraviolet treatment. The substance produced by strain GB-2 exhibited potential use in the areas of bio-control, food and medical application.
Anti-Bacterial Agents ; biosynthesis ; isolation & purification ; pharmacology ; Escherichia coli ; drug effects ; Fermentation ; Microbial Sensitivity Tests ; Seawater ; microbiology ; Staphylococcus aureus ; drug effects ; Streptomyces ; chemistry