Influence of DOC on the Inactivation Efficiency of Ozonation Assessed with Clostridium perfringens and a Lab-Scale Continuous Flow System

Routine quality monitoring for fecal indicators after ozonation at the river-lake waterworks Weesperkarspel of Amsterdam Water Supply (AWS) show large variation in inactivation. The influence of the high DOC in the water on the inactivation efficiency was investigated. Results showed a higher inactivation of Clostridium perfringens in the AWS water than in a water with low DOC at the same CT conditions. The contribution of the gas feed chamber to the overall inactivation of C. perfringens was high in the AWS water and was reduced after DOC reduction with GAC. This result may alter the current CT concept of the process. Further research will be focused on the ozone dosage strategy and control related to the required CT for inactivation and the production of by-products.     

Prevalence of Clostridium difficile in retailed meat in the Netherlands

Recent reports indicate that a large proportion of community-acquired Clostridium difficile infections (CA-CDI) are not linked to recent antibiotic therapy, older age, significant comorbidity or previous hospitalization. Possible community sources for CA-CDI include animals and food, and therefore a surveillance study on the prevalence of C. difficile in meat was performed. Samples of different meat species were collected from the retail trade and analyzed for the presence of C. difficile using a method that included selective enrichment in C. difficile broth, subsequent alcohol shock-treatment and plating onto C. difficile selective medium. C. difficile isolates were tested for the presence of toxin genes and were typed using PCR ribotyping. Of 500 samples tested, 8 (1.6%) were positive for the presence of C. difficile: 1 from lamb (6.3%) and 7 from chicken meat (2.7%). The isolated strains belonged to PCR ribotypes different from those that are currently most frequently found in patients with CDI in the Netherlands, except for C. difficile PCR ribotype 001 which was found in one chicken meat sample. This observation suggests that other matrices than meat may serve as a source for CA-CDI.     

Lag time variability in individual spores of Clostridium botulinum

Quantifying lag times from individual spores and the associated variability is an important part of understanding the hazard associated with spore-forming pathogens such as Clostridium botulinum. Knowledge of the underlying distribution would allow greater refinement of risk assessments. To date most studies have either examined lag time indirectly by measuring time to growth or have only examined the first stage of lag, germination. Recent studies have attempted to quantify the variability of spores during the different stages of lag phase and to examine the relationships between these stages. The effect of incubation temperature (22 °C, 15 °C, 10 °C or 8 °C), heat treatment (unheated or 80 °C for 20 s) and sodium chloride concentration in both the sporulation medium (0 or 3% w/v) or growth medium (0 or 2% w/v) on growth from individual spores has been examined. These studies found spores within a single population are very heterogeneous with large variability in all stages of lag. The duration and variability of times for germination, outgrowth and first doubling depended on both the historic treatment of the spores and the prevailing growth conditions, and the stage of lag most affected was treatment dependant.     

Clostridium butyricum Z-10的生长特性与培养基优化

对Clostridium butyricum Z 10的生长特性研究表明,其最适生长温度为36 ℃,最低和最高生长温度分别为16 ℃和44 ℃;最适起始生长pH值为7.2,最低和最高起始生长 pH值分别为4 6和10 6;最高耐胆酸盐质量浓度为4 g/dL,起始氧化还原电位为-21 mV;丁酸梭状芽孢杆菌对青霉素不敏感,主要代谢产物为丁酸和乙酸.通过对生长因子、氮源的选择性研究,得出采用酵母膏做为生长因子,胰蛋白胨作为氮源有较好的效果.优化培养基试验得出当胰蛋白胨质量浓度为22 g/L,葡萄糖质量浓度为 10 g/L,盐酸半胱氨酸质量浓度为 0 5 g/L时,总菌体浓度可达到3 6×108个/mL.     

几种香辛料精油对产气荚膜梭菌的体外抑菌效果

研究白芷精油、黑胡椒精油、茴香精油、肉桂精油、姜精油和艾草精油对产气荚膜梭菌（Clostridium perfringens,C.perfringens）的抑制作用,筛选出对产气荚膜梭菌有抑菌效果的香辛料精油。采用牛津杯法和两倍稀释法分别判定不同香辛料精油对C.perfringens（ATCC13124、CICC22949、C₁和C₂）的抑菌圈大小和最小抑菌浓度（Minimum inhibitory concentration,MIC）。结果显示:肉桂精油对C.perfringens的抑菌圈直径最大,且对产气荚膜梭菌ATCC13124、CICC22949、C₁和C₂的MIC分别为2.75、2.75、5.5、2.75 mg/m L;艾草精油对C.perfringens的MIC分别为11、2.75、11、2.75 mg/m L;黑胡椒精油对C.perfringens的MIC分别为22、11、11、5.5 mg/m L;茴香精油对C.perfringens的MIC分别为11、5.5、11、2.75 mg/m L;姜精油对C.perfringens的MIC分别为88、22、22、22 mg/m L。结论:除白芷精油以外,其他5种香辛料精油对C.perfringens均有不同程度的抑菌作用,其中肉桂精油对C.perfringens的抑菌效果最佳。      

一株高产丁酸的丁酸梭菌分离鉴定及其生物学性质研究

从健康成人新鲜粪便中筛选得到一株高产丁酸的丁酸梭菌C1-6,分析其生长特性和部分生物学性质。从形态学特征、生理生化试验、16S rDNA基因序列同源性以及多位点序列分型(Multi-site sequence typing,MLST)等方面对菌株进行鉴定;分析了该菌的部分生物学性质,包括发酵液中丁酸含量,对酸、胆盐、模拟消化道的耐受性以及抗生素抗性。结果表明,丁酸梭菌C1-6的24 h发酵液中丁酸含量达到2.1 g/L左右;对酸、胆盐以及模拟消化道环境具有较强的耐受性,其在pH2和pH3的酸性条件下孵育3 h后的存活率分别为72.5%和91.8%,对胆盐的耐受性随胆盐浓度的提高略有上升,当胆盐浓度为1%时,其存活率为104.63%,经过模拟消化道环境后其存活率仍高达93%以上;除对氯霉素、卡那霉素等氨基糖苷类抗生素和青霉素具有抗性外,对氨苄西林、四环素、万古霉素、头孢呋辛均比较敏感,有必要进一步评价其耐药基因的转移风险。由以上结果发现,丁酸梭菌C1-6有一定的潜在应用价值。     

Clostridiumnovyi’s Alpha-Toxin Changes Proteome and Phosphoproteome of HEp-2 Cells

C. novyi type A produces the alpha-toxin (TcnA) that belongs to the large clostridial glucosylating toxins (LCGTs) and is able to modify small GTPases by N-acetylglucosamination on conserved threonine residues. In contrast, other LCGTs including Clostridioides difficile toxin A and toxin B (TcdA; TcdB) modify small GTPases by mono-o-glucosylation. Both modifications inactivate the GTPases and cause strong effects on GTPase-dependent signal transduction pathways and the consequent reorganization of the actin cytoskeleton leading to cell rounding and finally cell death. However, the effect of TcnA on target cells is largely unexplored. Therefore, we performed a comprehensive screening approach of TcnA treated HEp-2 cells and analyzed their proteome and their phosphoproteome using LC-MS-based methods. With this data-dependent acquisition (DDA) approach, 5086 proteins and 9427 phosphosites could be identified and quantified. Of these, 35 proteins were found to be significantly altered after toxin treatment, and 1832 phosphosites were responsive to TcnA treatment. By analyzing the TcnA-induced proteomic effects of HEp-2 cells, 23 common signaling pathways were identified to be altered, including Actin Cytoskeleton Signaling, Epithelial Adherens Junction Signaling, and Signaling by Rho Family GTPases. All these pathways are also regulated after application of TcdA or TcdB of C. difficile. After TcnA treatment the regulation on phosphorylation level was much stronger compared to the proteome level, in terms of both strength of regulation and the number of regulated phosphosites. Interestingly, various signaling pathways such as Signaling by Rho Family GTPases or Integrin Signaling were activated on proteome level while being inhibited on phosphorylation level or vice versa as observed for the Role of BRCA1 in DNA Damage Response. ZIP kinase, as well as Calmodulin-dependent protein kinases IV & II, were observed as activated while Aurora-A kinase and CDK kinases tended to be inhibited in cells treated with TcnA based on their substrate regulation pattern.     

Antagonistic effect of fat on the antibotulinal activity of food preservatives and fatty acids

The effect of fat on the antibotulinal activity of 11 food preservatives, 12 free fatty acids, and nine lots of enzyme-modified cheese (EMC) was evaluated in a media system. Anhydrous milkfat or soybean oil was added to tubes of Trypticase–peptone–glucose–yeast extract medium (TPGY) supplemented with the additives (final pH adjusted to 5.9). Treatments were inoculated with 3-log₁₀ proteolytic Clostridium botulinum spores/ml (10-strain mixture of serotypes A and B) and incubated anaerobically at 30°C for up to 14 days. For the preservative and fatty acids studies, growth of C. botulinum was determined by measuring optical changes at OD_{640 nm}. Botulinal toxin production was determined in EMC-treatments using the mouse bioassay. Data revealed that the antibotulinal effects of nisin, and free fatty acids caprylic, capric, lauric, myristic, oleic, and linoleic acids were each significantly reduced in treatments supplemented with 20% fat (P<0.05). Similar trends were observed in TPGY supplemented with 20% fat and potassium sorbate, sorbic acid, monolaurin, polyphosphate emulsifier, or EDTA–lysozyme, but the differences were reduced. Fat was also antagonistic to the antibotulinal activity of five EMC-treatments. This study suggests that fat may reduce the efficacy of some antimicrobials added to or found naturally in foods.     

Evaluation of Ascorbic Acid as a Quorum-sensing Analogue to Control Growth, Sporulation, and Enterotoxin Production in Clostridium perfringens

ABSTRACT: Inhibition of quorum sensing measured by autoinducer-2 (AI-2) activity was investigated in the presence and absence of ascorbic acid, an AI-2 analogue. Subsequent effects on AI-2 production, as well as growth, sporulation, and enterotoxin ( C. perfringens enterotoxin [CPE]) production in Clostridium perfringens were examined. The addition of ascorbic acid to supernatants from ground beef resulted in a 100-fold decrease in AI-2 activity. The addition of sodium ascorbate, a nonacidic salt of ascorbic acid, also resulted in AI-2 assay inhibition. Spore production decreased in the presence of ascorbic acid. Western immunoblot analyses showed that CPE levels were highest after 24 h without ascorbic acid. This study explored the unique concept of signal inhibition to control pathogens in food.