BACKGROUND: The aim of this study was to develop and validate a multiplex real-time PCR assay for simultaneous identification and toxigenic type characterization of Clostridium difficile. METHODS: The multiplex real-time PCR assay targeted and simultaneously detected triose phosphate isomerase (tpi) and binary toxin (cdtA) genes, and toxin A (tcdA) and B (tcdB) genes in the first and sec tubes, respectively. The results of multiplex real-time PCR were compared to those of the BD GeneOhm Cdiff assay, targeting the tcdB gene alone. The toxigenic culture was used as the reference, where toxin genes were detected by multiplex real-time PCR. RESULTS: A total of 351 stool samples from consecutive patients were included in the study. Fifty-five stool samples (15.6%) were determined to be positive for the presence of C. difficile by using multiplex real-time PCR. Of these, 48 (87.2%) were toxigenic (46 tcdA and tcdB-positive, two positive for only tcdB) and 11 (22.9%) were cdtA-positive. The sensitivity, specificity, negative predictive value (NPV), and positive predictive value (PPV) of the multiplex real-time PCR compared with the toxigenic culture were 95.6%, 98.6%, 91.6%, and 99.3%, respectively. The analytical sensitivity of the multiplex real-time PCR assay was determined to be 103colonyforming unit (CFU)/g spiked stool sample and 0.0625 pg genomic DNA from culture. Analytical specificity determined by using 15 enteric and non-clostridial reference strains was 100%. CONCLUSIONS: The multiplex real-time PCR assay accurately detected C. difficile isolates from diarrheal stool samples and characterized its toxin genes in a single PCR run.
ADP Ribose Transferases/genetics ; Bacterial Proteins/*genetics ; Bacterial Toxins/*genetics ; Clostridium difficile/isolation & purification/*metabolism ; DNA, Bacterial/genetics/metabolism ; Enterotoxins/genetics ; Feces/*microbiology ; Humans ; Multiplex Polymerase Chain Reaction ; Prospective Studies ; Real-Time Polymerase Chain Reaction ; Triose-Phosphate Isomerase/genetics

BACKGROUND: Pseudomonas aeruginosa is a clinically important pathogen that causes opportunistic infections and nosocomial outbreaks. Recently, the type III secretion system (TTSS) has been shown to play an important role in the virulence of P. aeruginosa. ExoU, in particular, has the greatest impact on disease severity. We examined the relationship among the TTSS effector genotype (exoS and exoU), fluoroquinolone resistance, and target site mutations in 66 carbapenem-resistant P. aeruginosa strains. METHODS: Sixty-six carbapenem-resistant P. aeruginosa strains were collected from patients in a university hospital in Daejeon, Korea, from January 2008 to May 2012. Minimum inhibitory concentrations (MICs) of fluoroquinolones (ciprofloxacin and levofloxacin) were determined by using the agar dilution method. We used PCR and sequencing to determine the TTSS effector genotype and quinolone resistance-determining regions (QRDRs) of the respective target genes gyrA, gyrB, parC, and parE. RESULTS: A higher proportion of exoU+ strains were fluoroquinolone-resistant than exoS+ strains (93.2%, 41/44 vs. 45.0%, 9/20; P< or =0.0001). Additionally, exoU+ strains were more likely to carry combined mutations than exoS+ strains (97.6%, 40/41 vs. 70%, 7/10; P=0.021), and MIC increased as the number of active mutations increased. CONCLUSIONS: The recent overuse of fluoroquinolone has led to both increased resistance and enhanced virulence of carbapenem-resistant P. aeruginosa. These data indicate a specific relationship among exoU genotype, fluoroquinolone resistance, and resistance-conferring mutations.
ADP Ribose Transferases/genetics ; Anti-Bacterial Agents/*pharmacology ; Bacterial Proteins/genetics ; Bacterial Toxins/genetics ; Carbapenems/pharmacology ; Drug Resistance, Bacterial/*drug effects ; Fluoroquinolones/*pharmacology ; Genotype ; Humans ; Microbial Sensitivity Tests ; Multilocus Sequence Typing ; Mutation ; Pseudomonas aeruginosa/*genetics/isolation & purification/pathogenicity ; Sputum/microbiology ; Virulence

OBJECTIVETo investigate the effect of Pseudomonas quinolone signal (PQS) on the virulence of Pseudomonas aeruginosa.

METHODSPseudomonas aeruginosa strain PAO1 was treated with PQS alone, PQS plus farnesol, or farnesol alone. The transcriptional levels of the regulator gene ExsA and virulence protein gene ExoS of type III secretion system were examined using quantitative real-time PCR, and spectrophotometry was employed to detect pyocyanin production in the bacteria. The adhesion and invasiveness of the treated PAO1 in cultured alveolar epithelial cells A549 were assessed on plate count agar, and their effects on the survival of a mouse model of peritonitis was compared.

RESULTSThe increase or decrease of PQS did not affect the growth of PAO1. Compared with the untreated bacteria, PQS-treated PAO1 showed obviously increased transcription levels of ExsA and ExoS (P<0.01) and pyocyanin production, which was significantly lowered by farnesol (P<0.01). In A549 cell cultures, farnesol-treated PAO1 exhibited significantly lowered adhesion and invasiveness, while PQS-treated PAO1 caused a significantly decreased survival time of mice with peritonitis (P<0.01). Farnesol treatment did not obviously affected ExsA transcription (P>0.05) but caused a significant reduction in the transcriptional level of Exos (P<0.05) in PAO1. PQS showed no significant effect on the adhesion and invasiveness of PAO1 (P<0.05).

CONCLUSIONPQS can maintain the adhesion and invasiveness of Pseudomonas aeruginosa, and in the hosts of the bacteria, PQS concentration is positively correlated with pyocyanin production and hence negatively with the survival time of the hosts.

ADP Ribose Transferases ; genetics ; metabolism ; Animals ; Bacterial Adhesion ; Bacterial Proteins ; genetics ; metabolism ; Bacterial Toxins ; genetics ; metabolism ; Cell Line ; Humans ; Male ; Mice ; Mice, Inbred BALB C ; Peritonitis ; microbiology ; Pseudomonas aeruginosa ; genetics ; metabolism ; pathogenicity ; Quinolones ; pharmacology ; Recombinant Fusion Proteins ; genetics ; metabolism ; Signal Transduction ; Trans-Activators ; genetics ; metabolism ; Transcription, Genetic ; Virulence

OBJECTIVETo investigate drug-resistance and carriage of virulence factors of Pseudomonas aeruginosa (Pa) isolated from children.

METHODThirty-eight strains of Pa were collected and isolated in pediatric clinic during 2006-2009, and tests were undertaken to identify bacteria and susceptibility test was performed using VITEK-2 COMPACT GNI and AST-GN13 cards. The virulence factors were confirmed by using polymerase chain reaction (PCR) and sequencing.

RESULTAll the 38 strains of Pa were resistant to ampicillin, ampicillin/sulbactam, cefazolin, nitrofurantoin, trimethoprim/sulfamethoxazole, resistance rates were 100%. Except for ceftriaxone (60.53%), the resistance rates to other antibiotics were all below 16%. PCR test showed that all the 38 strains of Pa carried exotoxin A(toxA) and nitric oxide reductase A (norA), however, detective ratio of the other virulence factors, exoenzyme Y (exoY) was 84.21% (32/38), exoenzyme S (exoS) 57.89% (22/38), pyocyanin (pyp) 42.11% (16/38), exoenzyme U (exoU) 34.21% (13/38), and 38 strains of Pa did not carry exoenzyme T (exoT) and elastase B (lasB) without exception. By analyzing tests, we discovered that 3 pan-drug resistant strains of Pa were all combination of exo U+/pyp+, there were 4 strains of Pa which were moderately-resistant to imipenem, including exoU+/pyp+/exoY+ (2 isolates), exo U+/pyp+ (1 isolate), and exoY+/exoS+ (1 isolates). It indicated that the drug-resistance rate of exoU+/pyp+ is much higher, compared with exoS+ and exoY+. Molecular epidemiological detection revealed that 2 of 3 extensive-resistance strains of Pa were the same clone, but another one had 96.3% of homology with them.

CONCLUSIONThe above mentioned 34.21% of Pa isolated from children carried virulence factors toxA, norA, exoS, exoY, pyp and exoU. The strains with exoU/pyp had rather high resistance. The strains with pyp had strong toxicity, they easily cause generalized infection, the patients with them had very high mortality.

ADP Ribose Transferases ; genetics ; Bacterial Proteins ; genetics ; Bacterial Toxins ; genetics ; Carrier State ; epidemiology ; microbiology ; Child ; Drug Resistance, Multiple, Bacterial ; genetics ; Exotoxins ; Genes, Bacterial ; Humans ; Microbial Sensitivity Tests ; Polymerase Chain Reaction ; Pseudomonas Infections ; epidemiology ; genetics ; microbiology ; Pseudomonas aeruginosa ; genetics ; isolation & purification ; pathogenicity ; Virulence Factors ; genetics

OBJECTIVEThe aim of this study was to explore the possibility of creating a toxin, C-CPE-ETA', by fusing C-terminal high affinity binding domain of CPE (C-CPE) with a truncated form of Pseudomonas aeruginosa exotoxin A (ETA') and to examine whether C-CPE-ETA' could specifically target CLDN-3, 4 molecule and the targeted toxin was cytotoxic against CLDN-3,4-overexpressing ovarian cancer.

METHODSCLDN-3 and CLDN-4 expressions were analyzed at the mRNA level in three ovarian cancer cell lines and epithelial ovarian cancer tissues from 20 patients. After transforming an expression plasmid of C-CPE-ETA' into E. coli BL21 (DE3) plysS strain, the recombinant protein was purified using His-Bind resin chromatography column and analyzed by Western blot and Coomassie blue staining. The specific binding, proapoptotic and cytolytic activities were evaluated by flow cytometry, fluorescence microscopy with the JC-1 probe and MTT assay in CLDN-3,4-overexpressing ovarian cancer cells.

RESULTSQuantitive RT-PCR results showed there existed high levels of CLDN-3 and CLDN-4 in ovarian cancer cells, CAOV3, OVCAR3 and SKOV3. Moreover, high expressions of CLDN-3 and CLDN-4 were observed in 90.0% (18/20) and 60.0% (12/20) of ovarian cancer tissues, with an expression level 10-fold higher than that in the normal ovarian tissue. A 58 000 recombinant protein C-CPE-ETA' was demonstrated by Western blot and Coomassie blue staining. Purified and recombinant C-CPE-ETA' was bound with high affinity to CLDN-3,4-overexpressing ovarian cancer cells, CAOV3, OVCAR3 and SKOV3 cells. C-CPE-ETA' was strongly proapoptotic and cytotoxic towards the CLDN-3,4-overexpressing ovarian cancer cells. The concentration of IC(50) was 7.364 ng/ml for CAOV3 cells, 8.110 ng/ml for OVCAR3 cells and 22.340 ng/ml for SKOV3 cells, respectively. However, control CLDN-3,4-deficient cell line HUVEC was not susceptible to the recombinant C-CPE-ETA' at a concentration up to 10 µg/ml.

CONCLUSIONSThe C-CPE-ETA' protein exhibits remarkably specific cytotoxicity for CLDN-3,4-overexpressing ovarian cancer cells. Its therapeutic potential warrants further development for ovarian cancer molecular targeted therapy.

ADP Ribose Transferases ; metabolism ; physiology ; Apoptosis ; Bacterial Toxins ; metabolism ; Cell Line, Tumor ; Claudin-3 ; Claudin-4 ; Claudins ; genetics ; metabolism ; Enterotoxins ; metabolism ; physiology ; Exotoxins ; metabolism ; physiology ; Female ; Humans ; Immunotoxins ; metabolism ; Ovarian Neoplasms ; metabolism ; pathology ; RNA, Messenger ; metabolism ; Recombinant Fusion Proteins ; metabolism ; physiology ; Virulence Factors ; metabolism ; physiology

OBJECTIVETo study the clinical significance of virulence genes exo U and exo S of type III secretion system (TTSS) of Pseudomonas aeruginosa (PA).

METHODSOne hundred and eighty-nine clinical isolates of PA were collected from five hospitals. The incidence of virulence genes exo U and exo S in PA were determined with PCR. Minimum inhibitory concentration of anti-bacterial drug for PA was determined with microdilution method. The clinical features and outcomes of 60 hospitalized patients colonized or infected with exo U+/exo S- positive or exo U-/exo S+ positive PA isolated from sputum were analyzed retrospectively. Data were processed with chi-square test.

RESULTSAmong the 189 PA isolates, 85.2% (161/189) harbored TTSS genes, including exo U-/exo S+ type (120 isolates), exo U+/exo S- type (31 isolates), exo U-/exo S- type (7 isolates), and exo U+/exo S+ type (3 isolates). 72.0% (72/100) isolates from sputum and 81.5% (44/54) isolates from blood belonged to exo U-/exo S+ genotype. Compared with those of TTSS-negative isolates, the antimicrobial resistance of TTSS-positive isolates to cefoperazone/sulbactam, ceftazidime, amikacin, and cefepime were lower (with χ² value respectively 10.1, 16.1, 9.3, 33.8, P values all below 0.01). The antimicrobial resistance to all examined drug between exo U-/exo S+ type and exo U+/exo S- type isolates was close (with χ² values from 0.08 to 2.04, P values all above 0.05). Patients detected with exo U+/exo S- positive PA isolated from sputum were significantly associated with PA infection, and they usually had history of tracheal intubation, ICU hospitalization, and combined use of drugs for anti-infection treatment. Patients detected with exo U-/exo S+ positive PA isolated from sputum were significantly associated with PA colonization, which had basic lung disease and better outcome than the former infection type.

CONCLUSIONSThe TTSS exists in most clinical isolates of PA. Detection of exo U or exo S of PA isolated from sputum is helpful for the analysis of clinical features and outcome of patients.

ADP Ribose Transferases ; genetics ; metabolism ; Bacterial Proteins ; genetics ; metabolism ; Bacterial Secretion Systems ; genetics ; Bacterial Toxins ; genetics ; metabolism ; Drug Resistance, Bacterial ; Genes, Bacterial ; Humans ; Microbial Sensitivity Tests ; Pseudomonas Infections ; microbiology ; Pseudomonas aeruginosa ; genetics ; isolation & purification ; pathogenicity ; Retrospective Studies ; Virulence

M2 protein of type A influenza virus is a good candidate for universal influenza vaccine, exotoxin A of Pseudomonas aeruginosa may facilitate the immunogenicity of M2 protein. We constructed and expressed a prokaryotic expression plasmid containing a chimeric gene of M2 extracellular coding region and a partial PEA gene, and observed the immunoprotection in BALB/c mice vaccinated with the fusion protein. The fusion protein (ntPE-M2e) was generated by inserting the coding sequence of the M2e in place of Ib loop in PEA. This fusion protein was used to immunize BALB/c mice by subcutaneously injection with incomplete Freund's adjuvant and boost at weeks 3 and 7. The immunized mice were challenged with influenza virus strain A/PR/34/8. The fusion protein (ntPE-M2e) immunization protected mice against lethal viral challenge. ELISA and ELISPOT results demonstrated that the fusion protein could induce a strong systemic immune response against synthetic M2e peptide, and virus replication in the lungs of mice was inhibited in comparison with the control. This study provides foundation for developing broad-spectrum vaccines against type A influenza viruses.
ADP Ribose Transferases ; genetics ; Animals ; Bacterial Toxins ; genetics ; Enzyme-Linked Immunosorbent Assay ; Escherichia coli ; genetics ; Exotoxins ; genetics ; Female ; Gene Expression ; Immunization ; Influenza A virus ; immunology ; physiology ; Lung ; immunology ; virology ; Mice ; Mice, Inbred BALB C ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; immunology ; isolation & purification ; Viral Matrix Proteins ; biosynthesis ; genetics ; immunology ; isolation & purification ; Virulence Factors ; genetics

OBJECTIVETo investigate the pro-apoptotic effect of Her-2 targeted recombinant caspase-6 fusion protein on osteosarcoma SOSP-9607 cells.

METHODSRecombinant immunocasp-6 was generated by sequential fusion of the genes of a signal peptide, a single-chain Her-2 antibody (e23sFv), a PEA translocation domain (PEA aa253-364) and an active caspase-6. The immunocasp-6 gene was cloned into pCMV plasmid to construct a kind of eukaryotic expression vector, i.e. pCMV-e23sfv-PE II-caspase-6 (abbr. pCMV-6) and transfected into SOSP-9607 cells. Murine xenograft models were randomly divided into two groups that received i.m. injections of liposome encapsulated pCMV-6 or pCMV alone. The tumor volume and weight of the nude mice and the tumor weight of the cured mice were observed and statistically analyzed. The morphological changes of the tumors were examined with HE staining, apoptotic morphology of the tumor was observed by TUNEL staining and the gene expression was analyzed by immunohistochemical staining.

RESULTSThe tumor growth of the mice in the treatment group was significantly slower than that of the control group (P = 0.001). The weight of the nude mice in the treatment group was significantly higher than that of the control group (P = 0.0002). The tumor weight of the mice in the treatment group was significantly lower than that of the control group (P = 0.0006). HE and TUNEL staining of the tumor of nude mice in the treatment groups showed typical characteristics of apoptosis, while normal structure was found in the control group. Furthermore, caspase-6 was not found in the tumor and muscle tissues in the control group, but only in the treatment group by immunohistochemistry.

CONCLUSIONImmunocasp-6 can selectively recognize and bind to and kill HER-2 positive osteosarcoma cells, therefore, to offer some foundation for the clinical treatment of osteosarcoma.

ADP Ribose Transferases ; genetics ; Animals ; Apoptosis ; Bacterial Toxins ; genetics ; Bone Neoplasms ; metabolism ; pathology ; Caspase 6 ; genetics ; metabolism ; Cell Line, Tumor ; Exotoxins ; genetics ; Humans ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Neoplasm Transplantation ; Osteosarcoma ; metabolism ; pathology ; Plasmids ; Random Allocation ; Receptor, ErbB-2 ; genetics ; Recombinant Fusion Proteins ; genetics ; metabolism ; Transfection ; Tumor Burden ; Virulence Factors ; genetics

PURPOSE: Oral squamous carcinoma (OSCC) cells exhibit resistance to chemotherapeutic agent-mediated apoptosis in the late stage of malignancy. Increased levels of heat shock proteins 70 (HSP70) in cancer cells are known to confer resistance to apoptosis. Since recent advances in the understanding of bacterial toxins have produced new strategies for the treatment of cancers, we investigated the effect of Pseudomonas aeruginosa exotoxin A (PEA) on HSP70 expression and induction of apoptosis in chemoresistant OSCC cell line (YD-9). MATERIALS AND METHODS: The apoptotic effect of PEA on chemoresistant YD-9 cells was confirmed by MTT, Hoechst and TUNEL stains, DNA electrophoresis, and Western blot analysis. RESULTS: While YD-9 cells showed high resistance to chemotherapeutic agents such as etoposide and 5-fluorouraci (5-FU), HSP70 antisense oligonucelotides sensitized chemoresistant YD-9 cells to etoposide and 5-FU. On the other hand, PEA significantly decreased the viability of YD-9 cells by deteriorating the HSP70-relating protecting system through inhibition of HSP70 expression and inducing apoptosis in YD-9 cells. Apoptotic manifestations were evidenced by changes in nuclear morphology, generation of DNA fragmentation, and activation of caspases. While p53, p21, and E2F-1 were upregulated, cdk2 and cyclin B were downregulated by PEA treatment, suggesting that PEA caused cell cycle arrest at the G2/M checkpoint. CONCLUSION: Therefore, these results indicate that PEA reduced the chemoresistance through inhibition of HSP70 expression and also induced apoptosis in chemoresistant YD-9 cells.
ADP Ribose Transferases/*pharmacology ; Antineoplastic Agents/*pharmacology ; Apoptosis/drug effects ; Bacterial Toxins/*pharmacology ; Blotting, Western ; Carcinoma, Squamous Cell/drug therapy/*metabolism ; Cell Cycle/drug effects ; Cell Line, Tumor ; Chromatography, Liquid ; Cyclin B/metabolism ; Cyclin-Dependent Kinase 2/metabolism ; Drug Resistance, Neoplasm/*drug effects ; E2F1 Transcription Factor/metabolism ; Electrophoresis ; Exotoxins/*pharmacology ; HSP70 Heat-Shock Proteins/genetics/*metabolism ; Humans ; In Situ Nick-End Labeling ; Mouth Neoplasms/drug therapy/*metabolism ; Tandem Mass Spectrometry ; Tumor Suppressor Protein p53/metabolism ; Virulence Factors/*pharmacology

Pseudomonas aernginosa (PA) is one of the most universal pathogens in clinical diagnosis, and conventional detection assay has many disadvantages. In this research, a pair of specific primers and a TaqMan fluorescent probe were designed in the conservative region of ETA gene by the method of bioinformatics analysis, the detection method for PA was successfully developed. Different gradient concentrations of PA DNA and various pathogen DNA were amplified by fluorescence quantitative PCR (FQ-PCR) to confirm the specificity and sensitivity of the developed method. Results showed that the developed detection assay is more sensible and specific by comparison to the conventional FQ-PCR method, and it is valuable for research and application prospects.
ADP Ribose Transferases ; genetics ; Bacterial Toxins ; genetics ; DNA, Bacterial ; analysis ; Exotoxins ; genetics ; Fluorescent Dyes ; Fluorometry ; methods ; Polymerase Chain Reaction ; methods ; Pseudomonas aeruginosa ; genetics ; isolation & purification ; Sensitivity and Specificity ; Taq Polymerase ; Virulence Factors ; genetics