Analysis of common antigen of flagella in Bacillus cereus and Bacillus thuringiensis

Abstract Flagellar antigen of Bacillus cereus H.1 was purified and tested for serodiagnostic antigen by ELISA. The antibody against the flagellar antigen of B. cereus H.1 reacted not only with the homologous specific antigen but also reacted with the flagellar antigens of 23 strains of B. cereus . This common flagellar antigen of B. cereus was found to be due to 61-kDa protein by SDS-PAGE and immunoblot assay. Monoclonal antibody H15A5 against common antigenic epitope of B. cereus also reacted with flagellar antigens of 21 strains of Bacillus thuringiensis by ELISA. This monoclonal antibody reacted with the 61-kDa protein of the flagella of B. cereus H.1 and H.2 and B. thuringiensis Kurstaki HD1, Alesti and Aizawai juroi by immunoblot analysis. These results indicated that the common antigenic epitope of the 61-kDa protein existed in the flagella both of B. cereus and B. thuringiensis .     

我国若干地区蜡样芽孢杆菌723株噬菌体分型的初步报告

四年来,作者由水中获取蜡样芽孢杆菌噬菌体13株,旨在试作噬菌体分型。待检菌株共723株,由国内16个地区提供(其中食物中毒株121株;食品株602株)。分型结果显示:C29、C30、C24、C27、C19和A_4等型别占优势。中毒株     

Presence of calmodulin-like calcium-binding protein in Bacillus cereus T spores

Abstract Bacillus cereus T spores were extensively washed, broken, and heated at 90°C for 2 min. Using calcium-dependent hydrophobic interaction chromatography, a single peak protein fraction was obtained which possessed calcium-binding capacity and some characteristics of calmodulin. This heat-stable protein fraction was retained by hydrophobic matrices (Phenyl-Sepharose) or a calmodulin antagonist (naphthalenesulfonamide) in a calcium-dependent manner. Calcium binding ability was verified by ⁴⁵Ca autoradiography and a competitive calcium binding assay using Chelex-100. The crude spore extract displaced bovine brain calmodulin from its antibody in a radioimmunoassay and the immunoreactive specific activity of the partially purified fraction was approx. 200-fold greater than the crude spore extract. Thus, B. cereus T spores have a calcium-binding protein with calmodulin-like properties.     

电脉冲穿孔法将苏云金杆菌δ-内毒素基因导入野生型芽孢杆菌

利用电脉冲穿孔法将带有苏云金杆菌毒蛋白基因的穿梭质粒导入几株野生型芽孢杆菌中。它们是野生型的蜡状芽孢杆菌、短芽孢杆菌和枯草芽孢杆菌。通过观察在新霉素和氨苄青霉素平板上长出的抗性菌落数,计算出转化效率为10~1—10~4转化子/μgDNA。从转化子中分离到的质粒DNA大小及其用HindⅢ酶切的片段与原始质粒DNA相同,毒性测试表明重组转化子对烟青虫六天的致死率达90—100％。     

青霉素酶发酵液的预处理和酶学特性

研究了在发酵液中添加絮凝剂对发酵液进行预处理,对蜡状芽孢杆菌（Bacillus cereus）产生青霉素酶的影响与酶学特性。实验结果表明,发酵液膜处理的难易程度与芽孢释放程度成正相关,在发酵液中添加0．02g／mL自制絮凝剂进行预处理,过滤效率提高10倍,而酶活损失仅5％;酶学特性研究结果表明,该酶最佳反应温度55℃时,酶的最适反应pH值7．0,金属离子锌、锰、镁对酶有激活作用,其浓度为0．25moL／L;酶热稳定性研究结果表明,在75℃下保温30min时,酶活损失85％。该酶在过量青霉素底物下,酶促反应为0级反应。     

Bacillus cereus biofilm formation on central venous catheters of hospitalised cardiac patients

Formation of bacterial biofilms is a risk with many in situ medical devices. Biofilm-forming Bacillus species are associated with potentially life-threatening catheter-related blood stream infections in immunocompromised patients. Here, bacteria were isolated from biofilm-like structures within the lumen of central venous catheters (CVCs) from two patients admitted to cardiac hospital wards. Isolates belonged to the Bacillus cereus group, exhibited strong biofilm formation propensity, and mapped phylogenetically close to the B. cereus emetic cluster. Together, whole genome sequencing and quantitative PCR confirmed that the isolates constituted the same strain and possessed a range of genes important for and up-regulated during biofilm formation. Antimicrobial susceptibility testing demonstrated resistance to trimethoprim-sulphamethoxazole, clindamycin, penicillin and ampicillin. Inspection of the genome revealed several chromosomal β-lactamase genes and a sulphonamide resistant variant of folP. This study clearly shows that B. cereus persisting in hospital ward environments may constitute a risk factor from repeated contamination of CVCs.     

Evidence for non-ribosomal peptide synthetase production of cereulide (the emetic toxin) in Bacillus cereus

Little is known about the process whereby the emetic toxin (or cereulide) of Bacillus cereus is produced. Two cereulide-producing strains of B. cereus were cloned and sequenced following polymerase chain reaction (PCR) amplification with primers that were specific for conserved regions of non-ribosomal peptide synthetase (NRPS) genes. The cloned regions of the B. cereus strains were highly homologous to conserved regions of other peptide synthetase nucleotide sequences. Primers were designed for two variable regions of the NRPS gene sequence to ensure specificity for the emetic strains. A total of 86 B. cereus strains of known emetic or non-emetic activity were screened using these primers. All of the emetic strains (n=30) displayed a 188 bp band following amplification and gel electrophoresis. We have developed an improved method of identifying emetic strains of B. cereus and provided evidence that cereulide is produced by peptide synthetases.     

Conjugative transfer, stability and expression of a plasmid encoding a cry1Ac gene in Bacillus cereus group strains

The plasmid pHT73 containing cry1Ac and tagged with an erythromycin resistance gene was transferred from Bacillus thuringiensis subspecies kurstaki KT0 to several Bacillus cereus group strains by conjugation. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and phase contrast microscopy showed that the transconjugants containing plasmid pHT73 could express Cry1Ac toxin and produce bipyramidal crystalline inclusion bodies during sporulation. The study demonstrated that pHT73 could be transferred to B. thuringiensis subsp. kurstaki, several B. cereus strains and Bacillus mycoides. Under non-selective conditions, the stability of the pHT73 plasmid in the transconjugants was found to be 58.2-100% after 100 generations and 4-96% after 200 generations. The variations are mainly caused by the choice of receptor strain.     

Novel method for the proteomic investigation of a dairy-associated Bacillus cereus biofilm

The biofilm proteome of a dairy-associated Bacillus cereus strain (B. cereus 5) was investigated. Biofilm biomass of sufficient concentration for 2D-PAGE was obtained by growing the culture in the presence of glass wool. B. cereus 5 readily attached to the glass wool and biofilms formed within 18 h. The biofilm proteome of whole-cell proteins revealed that 10 proteins were synthesized as a result of surface attachment of which four were unique to the biofilm profile. Seven proteins appeared to be absent in the biofilm profile. The altered proteomes indicated that changes took place in the regulation of protein expression when B. cereus 5 cells attached to surfaces.