The entomopathogenic bacterium, Xenorhabdus nematophila was isolated from the hemolymph of Galleria mellonella infected with Steinernema carpocapsae. The bacterial cells and its metabolic secretions have been found lethal to the Galleria larvae. Toxic secretion in broth caused 95% mortality within 4 d of application whereas the bacterial cells caused 93% mortality after 6 d. When filter and sand substrates were compared, the later one was observed as appropriate. Similarly, bacterial cells and secretion in broth were more effective at 14% moisture and 25 degrees C temperature treatments. Maximum insect mortality (100%) was observed when bacterial concentration of 4x10(6) cells/ml was used. Similarly, maximum bacterial cells in broth (95%) were penetrated into the insect body within 2 h of their application. However, when stored bacterial toxic secretion was applied to the insects its efficacy declined. On the other hand, when the same toxic secretion was dried and then dissolved either in broth or water was proved to be effective. The present study showed that the bacterium, X. nematophila or its toxic secretion can be used as an important component of integrated pest management against Galleria.
Animals ; Bacterial Proteins ; pharmacology ; Bacterial Toxins ; pharmacology ; Larva ; drug effects ; microbiology ; Moths ; drug effects ; microbiology ; Nematoda ; microbiology ; Pest Control, Biological ; methods ; Survival Analysis ; Survival Rate ; Xenorhabdus ; metabolism ; pathogenicity

We evaluated the new C. DIFF QUIK CHEK COMPLETE (CD COMPLETE; TechLab, USA), which is a rapid membrane enzyme immunoassay that uses a combination of glutamate dehydrogenase (GDH) antigen and toxin A and B detection. A total of 608 consecutive loose stool specimens collected from the patients with suspected Clostridium difficile infection (CDI) from August to December 2012 were subjected to the CD COMPLETE and VIDAS Clostridium difficile A & B (VIDAS CDAB; bioMerieux, France). Their performances were compared with a toxigenic culture as a reference. Stool specimens that were culture-negative and CD COMPLETE- or VIDAS CDAB-positive were analyzed by using an enrichment procedure. In comparison to the toxigenic cultures, sensitivity, specificity, positive predictive values (PPV), and negative predictive values (NPV) were 63.6%, 98.0%, 76.1%, and 96.4%, respectively, for the CD COMPLETE-toxin and 75.5%, 97.4%, 72.5%, and 97.8%, respectively, for the VIDAS CDAB. In comparison to the enriched C. difficile cultures, the sensitivity, specificity, PPV, and NPV for the CD COMPLETE-GDH were 91.0%, 92.4%, 70.5%, and 98.1%, respectively. The CD COMPLETE is a reliable method for the diagnosis of CDI and provides greater sensitivity than toxin enzyme immunoassay alone. Furthermore, the CD COMPLETE-GDH has advantages over direct culture in detecting C. difficile.
Bacterial Proteins/*analysis ; Bacterial Toxins/*analysis ; Clostridium Infections/*diagnosis/microbiology ; Clostridium difficile/enzymology/*isolation & purification/metabolism ; Enterotoxins/*analysis ; Feces/microbiology ; Glutamate Dehydrogenase/*analysis ; Humans ; *Immunoenzyme Techniques ; Reagent Kits, Diagnostic ; Sensitivity and Specificity

BACKGROUND: Enzyme immunoassay (EIA) capable of detecting both toxin A and toxin B is strongly recommended for the diagnosis of Clostridium difficile associated disease. Therefore, we evaluated two different EIAs for the detection of C. difficile toxin A/B. METHODS: For a total of 228 stool specimens we performed bacteriologic cultures for C. difficile and examined for toxin A and toxin B using enzyme linked fluorescent immunoassay (ELFA; VIDAS CDAB, Bio-Merieux sa, France) and ELISA (C.DIFFICILE TOX A/B II, TECHLAB, USA). We also performed PCR assays for toxin A and B genes in 117 C. difficile isolates that grew from the stool cultures and compared the results with those obtained with the two different EIAs. RESULTS: The concordance rate between ELFA and ELISA was 85.5% (195/228). Using the culture and PCR results as the standard, the sensitivity/specificity of the ELFA and ELISA were 65.0%/72.1% and 71.8%/70.3%, and for positive/negative predictive values were 78.4%/69.6% and 71.8%/70.3%, respectively (P value >0.05). No differences were observed between the results of ELFA and ELISA with toxin A- toxin B+ strains of C. difficile. CONCLUSIONS: The sensitivity of the ELISA was slightly higher than that of ELFA for toxin A and toxin B, but the specificity and positive predictive value of the ELFA were rather higher than those of the ELISA, although no statistical differences were observed. A bacteriologic culture and PCR assay for toxin genes are recommended in case the both EIAs are negative.
Bacterial Proteins/*analysis/genetics/immunology ; Bacterial Toxins/*analysis/genetics/immunology ; Clostridium difficile/genetics/isolation & purification/*metabolism ; Enterotoxins/*analysis/genetics/immunology ; Enzyme-Linked Immunosorbent Assay/*methods ; Feces/microbiology ; Fluorescent Dyes/chemistry ; Humans ; Reagent Kits, Diagnostic

OBJECTIVETo clone the recombinant fusion gene of Escherichia coli heat-liable enterotoxin B subunit (Ltb) and Actinobacillus actinomycetemcomitans fimbria associative protein (Fap).

METHODSTwo couples of primers were designed for PCR according to the known sequence of ltb and fap. The ltb and fap gene were obtained by amplification PCR technique from plasmid EWD299 of Escherichia coli and Actinobacillus actinomycetemcomitans 310 DNA respectively, and fused them by PCR. The fusion gene ltb-fap were cloning into plasmid pET28a(+). The recombined plasmid pET28a ltb-fap was transformed into Escherichia coli DH5alpha. The recombinant was screened and identified by restriction enzyme and PCR. The cloned gene was sequenced.

RESULTSThe ltb-fap about 531bp in size was obtained successfully, and identified by PCR, restrictive enzyme and sequence analysis.

CONCLUSIONThe vector of pET28a ltb-fap was obtained.

Aggregatibacter actinomycetemcomitans ; Bacterial Toxins ; Cloning, Molecular ; Cloning, Organism ; Enterotoxins ; Escherichia coli ; Escherichia coli Proteins ; Hot Temperature ; Plasmids ; Polymerase Chain Reaction ; Recombinant Fusion Proteins ; Sequence Analysis

BACKGROUND: We evaluated the performance of four commercial nucleic acid amplification tests (NAATs: Xpert C. difficile, BD MAX Cdiff, IMDx C. difficile for Abbott m2000, and Illumigene C. difficile) for direct and rapid detection of Clostridium difficile toxin genes. METHODS: We compared four NAATs on the same set of 339 stool specimens (303 prospective and 36 retrospective specimens) with toxigenic culture (TC). RESULTS: Concordance rate among four NAATs was 90.3% (306/339). Based on TC results, the sensitivity and specificity were 90.0% and 92.9% for Xpert; 86.3% and 89.3% for Max; 84.3% and 94.4% for IMDx; and 82.4% and 93.7% for Illumigene, respectively. For 306 concordant cases, there were 11 TC-negative/NAATs co-positive cases and 6 TC-positive/NAATs co-negative cases. Among 33 discordant cases, 18 were only single positive in each NAAT (Xpert, 1; Max, 12; IMDx, 1; Illumigene, 4). Positivity rates of the four NAATs were associated with those of semi-quantitative cultures, which were maximized in grade 3 (>100 colony-forming unit [CFU]) compared with grade 1 (<10 CFU). CONCLUSIONS: Commercial NAATs may be rapid and reliable methods for direct detection of tcdA and/or tcdB in stool specimens compared with TC. Some differences in the sensitivity of the NAATs may partly depend on the number of toxigenic C. difficile in stool specimens.
Bacterial Proteins/genetics ; Bacterial Toxins/genetics ; Clostridium Infections/*diagnosis/microbiology ; Clostridium difficile/*genetics/isolation & purification ; DNA, Bacterial/*analysis/metabolism ; Enterotoxins/genetics ; Feces/*microbiology ; Humans ; *Multiplex Polymerase Chain Reaction ; Reagent Kits, Diagnostic ; Sensitivity and Specificity

BACKGROUND: ChromID Clostridium difficile agar (IDCd; bioMerieux SA, France) is a recently developed chromogenic medium for rapid and specific isolation of C. difficile. We compared the performance of IDCd with that of Clostridium difficile Selective Agar (CDSA). METHODS: A total of 530 fresh stool specimens were collected from patients with clinical signs compatible with C. difficile infection, and cultures for C. difficile were performed on IDCd and CDSA. C. difficile colonies were identified by spore staining, odor, use of an ANI identification test kit (bioMerieux SA), and multiplex PCR for tcdA, tcdB, and tpi. RESULTS: The concordance rate between IDCd and CDSA was 90.6% (480/530). The positivity rates on IDCd on days 1 and 2 (55.6% and 85.0%, respectively) were significantly higher than those on CDSA (19.4% and 75.6%, respectively) (P<0.001 for day 1 and P=0.02 for day 2), but the detection rates on IDCd and CDSA on day 3 were not different (89.4% vs. 82.8%, P=0.0914). On day 3, the recovery rates for non-C. difficile isolates on IDCd and CDSA were 30.2% (160/530) and 22.1% (117/530), respectively (P=0.0075). Clostridium spp. other than C. difficile were the most prevalent non-C. difficile isolates on both media. CONCLUSIONS: The culture positivity rates on IDCd and CDSA were not different on day 3 but IDCd may allow for rapid and sensitive detection of C. difficile within 2 days of cultivation.
Agar/*chemistry ; Bacterial Proteins/genetics ; Bacterial Toxins/genetics ; Clostridium difficile/genetics/*isolation & purification ; DNA, Bacterial/analysis ; Enterocolitis, Pseudomembranous/diagnosis/microbiology ; Enterotoxins/genetics ; Feces/*microbiology ; Humans ; Multiplex Polymerase Chain Reaction ; Reagent Kits, Diagnostic ; Triose-Phosphate Isomerase/genetics

OBJECTIVETo evaluate the impacts of Helicobacter pylori (H. pylori) infection on atherosclerosis and plasma lipid levels in high-cholesterol diet fed C57BL/6 mice.

METHODFemale C57BL/6 mice were randomly divided into 4 groups (n = 12 each): fed with normal chow diet (A), infected with H. pylori (B), fed with high-cholesterol diet (C) and infected with H. pylori and fed with high-cholesterol diet (D). After 52 weeks, plasma levels of lipids were measured and aortic atherosclerosis was observed. The ureA, ureC, cagA and vacA DNA were also detected by PCR in the aortic arteries.

RESULT(1) Prevalence of atherosclerosis was similar between group C and D (91.6% vs. 100%, P > 0.05) while there was no atherosclerosis in group A and B. H. pylori infected mice showed more obvious inflammation in gastric mucosa than mice without H. pylori infection. (2) The plasma levels of triglyceride, total cholesterol and LDL were higher and HDL was lower in group B, C and D than those in group A and in group D than in group C (all P < 0.05). (3) Roberts & Thompson scores and number of foam cells in plaques were significantly higher in group D compared with those in group C (all P < 0.05). (4) ureC DNA was detected in 5 out of 12 aortic arteries of mice in group D but not in group A, B and C.

CONCLUSIONOur results suggested that H. pylori infection might enhance the atherosclerotic lesion formation in this mouse model.

Animals ; Atherosclerosis ; microbiology ; pathology ; Bacterial Proteins ; genetics ; Bacterial Toxins ; genetics ; Cholesterol ; blood ; Cholesterol, Dietary ; adverse effects ; DNA, Bacterial ; analysis ; Female ; Helicobacter Infections ; pathology ; Helicobacter pylori ; genetics ; Mice ; Mice, Inbred C57BL ; Triglycerides ; blood ; Urease ; genetics

OBJECTIVETo investigate the immunoregulatory effects of pertussis protein on airway inflammatory, IFN-gamma/IL-4 ratio in bronchoalveolar lavage fluids(BALF) and airway hyperresponsiveness (AHR) in the sensitized mice.

METHODSThe sensitized mice were reexposed to ovalbumin and the airway response to methacholine injection was monitored. Inflammatory cells and cytokines IFN-gamma/IL-4 ratio in BALF were measured. Lung tissue specimens were collected for histological examination.

RESULTIntramuscular injection or intranasal instillation of pertussis protein inhibited changes in lung resistance and lung dynamic compliance, upregulated IFN-gamma/IL-4 ratio and decreased eosinophil accumulation in a dose-dependent manner. Pathological examination showed that goblet cell hyperplasia and inflammatory cells infiltration in lung tissue were suppressed by pertussis protein.

CONCLUSIONPertussis protein inhibits the inflammation and regulates the function of lungs in asthma mice, suggesting its potential application in treatment of asthma.

Albumins ; Animals ; Asthma ; chemically induced ; immunology ; therapy ; Bacterial Proteins ; immunology ; pharmacology ; Bacterial Toxins ; immunology ; pharmacology ; Bronchoalveolar Lavage Fluid ; chemistry ; Interferon-gamma ; analysis ; Interleukin-4 ; analysis ; Male ; Methacholine Chloride ; Mice ; Mice, Inbred ICR

OBJECTIVETo identify and determine the congener and level of microcystins in the source water of Taihu Lake.

METHODSImproved method of SPE combined with HPLC was employed to detect the concentration and varieties of microcystins in source water and bloom samples collected from Meiliang Bay, Taihu Lake.

RESULTSThe contents of two predominant microcystin components, MC-RR, and MC-LR, were relatively high in samples during warm months and correlated with the phase of algae growth. The maximum concentrations of MC-RR and MC-LR in water sample reached 3.09 +/- 0.53 microg/L and 2.39 +/- 0.41 microg/L during the period of water bloom in September 2004, respectively. Even without waterbloom, the concentration of MC-LR in source water sample was still higher than the guideline value.

CONCLUSIONThe status of microcystin pollution in this region is serious and measures to monitor and control the growth of cyanobacteria are urgently needed.

Animals ; Bacterial Toxins ; analysis ; China ; Chromatography, High Pressure Liquid ; Cyanobacteria ; growth & development ; isolation & purification ; Environmental Monitoring ; Fresh Water ; analysis ; chemistry ; Microcystins ; analysis ; chemistry ; Spectrophotometry, Ultraviolet ; Time Factors ; Water Microbiology ; Water Pollutants, Chemical

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