基于全基因组测序技术的食品分离致泻大肠埃希菌的分子特征及耐药性研究

目的 了解上海市肉品和水产品中分离致泻大肠埃希菌（DEC）的生物学特征及耐药性,为防控由该菌引起的食源性疾病提供科学依据。方法对食品分离DEC菌株进行全基因组测序及药物敏感性试验,利用BioNumerics软件对全基因组测序数据进行拼接,利用拼接序列开展多位点序列分型、毒力基因和耐药基因分析。结果本研究中56株DEC菌株中肠道聚集性大肠埃希菌（EAEC）占比最高,达73.2%,且以astA基因为主（90.2%）。56株DEC分为37个ST型,显示高度多样性。DEC菌株对喹诺酮类抗生素耐药性较高（64.3%）,其次是对氨基糖苷类、β-内酰胺类抗生素和四环素类抗生素耐药,且多重耐药性菌株占48.2%,耐药基因相应的以喹诺酮类、氨基糖苷类、β-内酰胺类抗生素耐药基因为主,且大多数对抗生素耐药的菌株均携带其相应的耐药基因。结论 EAEC是上海市食品中分离DEC菌株的主要型别,对喹诺酮类、β-内酰胺类、氨基糖苷类抗生素的耐药率较高,且携带相应的耐药基因,全基因组测序技术可应用于DEC的分子生物学监测中,为疾病预防控制提供科学依据。     

Short communication: the complete genome sequence of Bifidobacterium animalis subspecies animalis ATCC 25527(T) and comparative analysis of growth in milk with B. animalis subspecies lactis DSM 10140(T)

The objective of this work was to sequence the genome of Bifidobacterium animalis ssp. animalis ATCC 25527^T, the subspecies most closely related to B. animalis ssp. lactis, some strains of which are widely added to dairy foods as probiotics. The complete 1,932,963-bp genome was determined by a combination of 454-shotgun sequencing and PCR gap closing, and the completed assembly was verified by comparison with a KpnI optical map. Comparative analysis of the B. animalis ssp. animalis ATCC 25527^T and B. animalis ssp. lactis DSM 10140^T genomes revealed high degrees of synteny and sequence homology. Comparative genomic analysis revealed 156 and 182 genes that were unique to and absent in the B. animalis ssp. animalis genome, respectively. Among these was a set of unique clustered regularly interspaced short palindromic repeats (CRISPR)-associated genes and a novel CRISPR locus containing 30 spacers in the genome of B. animalis ssp. animalis. Although previous researchers have suggested that one of the defining phenotypic differences between B. animalis ssp. animalis and B. animalis ssp. lactis is the ability of the latter to grow in milk and milk-based media, the differential gene content did not provide insights to explain these differences. Furthermore, growth and acid production in milk and milk-based media did not differ significantly between B. animalis ssp. lactis (DSM 10140^T and Bl04) and B. animalis ssp. animalis (ATCC 25527^T). Growth of these strains in supplemented milk suggested that growth was limited by a lack of available low-molecular-weight nitrogen in the 3 strains examined.     

Effects of BmKIT3R gene transfer on the development and survival of silkworm Bombyx mori

To verify the effects of gain-of-function mutation of the BmKIT3^R gene (from the Chinese scorpion Buthus martensii Karsch) on the development and survival rate of insects and to explore a novel strategy for pest control, the effects of BmKIT3^R gene transfer on the development and survival rate of silkworms were investigated. A novel transgenic vector derived from the piggyBac transposon with the BmKIT3^R gene controlled by the Bmhsp20.4 promoter was transferred into silkworm eggs. Transgenic silkworms were obtained after screening with GFP and G418 antibiotics and verification by PCR and dot hybridization. The results showed that the oviposition number decreased by 18.9%, and the hatching and final survival rates were approximately 63% and 47.5%, respectively. Some 18.9% of surviving pupae died before developing into moths in the G3 generation. A specific band corresponding to BmKIT3^R was detected for transgenic silkworms by Western blotting. This indicates that the Bmhsp 20.4 promoter has constitutive expression activity. The significant decrease in the survival rate suggests that pest population numbers could be effectively controlled by using BmKIT3^R gene transfer. Furthermore, it can be speculated that pupal development to moths could be blocked if BmKIT3^R were specially expressed in the pupal stage and reeling with fresh cocoons was performed.     

Complete genome sequence and characterization of the N-acylhomoserine lactone-degrading gene of the potato leaf-associated Solibacillus silvestris

N-Acylhomoserine lactones (AHLs) are used as quorum-sensing signal molecules by many gram-negative bacteria. We have reported that Solibacillus silvestris, which was isolated from the potato leaf, has AHL-degrading activity. To identify the AHL-degrading gene, whole genome sequencing of S. silvestris StLB046 was performed by using pyrosequencing technology. As the result of the BLAST search, one predicted ORF (ahlS) showed slight similarity to AiiA-like AHL lactonase from Bacillus cereus group. Escherichia coli harboring the ahlS-expressing plasmid showed high AHL-degrading activity. The ahlS-cording region was also amplified by PCR from the other potato leaf-associated and AHL-degrading S. silvestris strains. Purified AhlS as a maltose binding fusion protein showed high AHL-degrading activity and catalyzes AHL ring opening by hydrolyzing lactones. In addition, expression of ahlS in plant pathogen Pectobacterium carotovorum subsp. carotovorum attenuated maceration of the potato slices. Our results suggest that AHL-degrading activity of ahlS might perform useful functions such as useful biocontrol agents.     

Comparative Study on the Physical Properties of Edgeworthia and Wikstroemia Handmade Paper

Both Edgeworthia and Wikstroemia have been used traditionally in hand papermaking in China. As plants from the Thymelaeaceae family, these species have been mentioned periodically and have been mixed or substituted with each other. This study reviewed their papermaking background and status, and selected one of the Edgeworthia and three of the Wikstroemia raw materials that are still in use. Pulp was cooked under the same conditions in the laboratory, and handsheets of pure and mixed materials were obtained using a Rapid-K?then sheet former. A series of physical properties, such as color, structure, and mechanical properties were tested, followed by a comparison and discussion of the raw material properties. The results show that Wikstroemia fibers are generally thinner and shorter, but there are variations between the different genera of raw materials. Thus, compared with Edgeworthia, Wikstroemia has some advantages in tensile strength, although it shows some disadvantages when it comes to tearing strength. The performance of a mixed-ingredient paper sheet falls between the performance of two pure-component papers. For cost and performance reasons, adding a small amount of Wikstroemia (e.g., <20%) to Edgeworthia is preferable.     

Evaluation of the effect of defrosting practices of ground beef on the heat tolerance of Listeria monocytogenes and Salmonella Enteritidis

The effect of common defrosting practices of ground beef, including (i) defrosting in the refrigerator (5 °C for 15 h), (ii) defrosting at room temperature (25 °C for 12 h) and (iii) defrosting in the microwave, on the heat tolerance of artificially inoculated Listeria monocytogenes and Salmonella Enteritidis, was studied. The thermal inactivation of S. Enteritidis was not, overall, affected by defrosting practices. In contrast, defrosting at room temperature resulted, overall, in an increased heat tolerance of L. monocytogenes compared to the rest tested defrosting practices. Inactivation kinetics of the two pathogens for the different defrosting practices were determined by fitting the data to the Weibull model. The δ parameter of the Weibull model (heat challenge time (min) required for the first 1-log reduction) for S. Enteritidis and for defrosting at 25 °C, microwave defrosting, defrosting at 5 °C and for the control (fresh ground beef inoculated with the pathogens just before the heat challenge trials) was 1.13, 1.62, 1.60 and 0.96, respectively, while the corresponding values for L. monocytogenes were 20.13, 10.82, 9.95 and 9.47, respectively. The findings of this study should be useful in risk assessments and in developing food handling guidelines for the consumers.     

部分瑞香科植物韧皮部化学组分与纤维形态研究

为评价几种瑞香科植物的纤维品质,分析了不同地区北江荛花、了哥王、多毛荛花、细轴荛花和狼毒2～3年生枝条韧皮或根皮纤维化学组分、纤维形态和细胞壁超微结构。结果表明,(1)不同地区北江荛花（64.25%～72.31%）、肇庆细轴荛花（71.33%）、多毛荛花（65.6%）和狼毒（59.04%）综纤维素含量均高于或接近优质传统手工纸原料檀皮（61.43%）和构皮（67.50%）,而木质素含量均低于檀皮和构皮;(2)融水北江荛花纤维最长（5.51 mm）,其次为遂昌北江荛花、肇庆北江荛花和狼毒,分别为5.40、5.09和5.05 mm,而茂名北江荛花纤维最短,仅为3.09 mm。融水北江荛花、肇庆北江荛花纤维长宽比较大,分别为565.2、538.4;(3)超微结构显示,城步北江荛花、融水北江荛花、遂昌北江荛花、肇庆北江荛花以及多毛荛花韧皮纤维细胞壁较薄,而茂名北江荛花、肇庆细轴荛花、狼毒和肇庆了哥王韧皮纤维细胞壁较厚。     

Influence of fruit ripeness and salt concentration on the microbial processing of Arbequina table olives

Arbequina table olives are processed as “naturally green olives”, they are directly placed in brine and fermentation starts spontaneously. Olives are harvested just before they change to ‘turning colour’. Different salt concentrations are used depending on the producer. The aim of the study was to evaluate how (i) the ripeness of the olive when it is harvested and (ii) the salt concentration of the brine influence the different microorganism populations in brine during the fermentation of Arbequina table olives.The results showed that the Enterobacteriaceae population lasted longer in black and turning colour olives than in green olives, whereas the growth of lactic acid bacteria was delayed in green olives. A higher salt concentration favoured the elimination of Enterobacteriaceae and hindered yeast growth. The main yeast species identified were Pichia anomala, Candida sorbosa and Candida boidinii, while Lactobacillus plantarum was the only lactic acid bacteria species involved in the process. In a sensory test, panellists preferred green olives and were not able to tell the laboratory-scale processed olives from a commercial sample, nor could they distinguish green olives from different brines.     

Effect of Zataria multiflora Boiss. essential oil and nisin on Salmonella typhimurium and Staphylococcus aureus in a food model system and on the bacterial cell membranes

The effects of different concentrations of Zataria multiflora Boiss. essential oil (EO: 0, 5, 15 and 30 μl 100 ml⁻¹) and nisin (N: 0, 0.25 and 0.5 μg ml⁻¹), temperatures (T: 25 and 8 °C), and storage times (up to 21 days) on growth of Salmonella typhimurium and Staphylococcus aureus in a commercial barley soup were evaluated in a factorial design study. The growth of S. typhimurium was significantly (P < 0.05) decreased by EO concentrations and their combinations with N concentrations at 8 °C. For S. aureus, the viable count was significantly (P < 0.05) inhibited by EO and N concentrations and their combinations, incubated at both storage temperatures. The mechanism of the antimicrobial action of EO, N, and their combinations against cell membranes of the tested organisms were also studied by measurement of the release of cell constituents and by the electronic microscopy observations of the cells. The significant increase of the cell constituents’ release of both organisms was observed as a result of treatments with EO and EO in combination with N. Electronic microscopy observations revealed that the cell membranes of S. typhimurium treated by EO and EO in combination with N were significantly damaged, while cells treated with only N looked similar to untreated cells. The electron micrographs of treated cells of S. aureus with EO, N, and their combination also showed important morphological damages and disrupted membranes.     

基于毒力基因组合评价我国产志贺毒素大肠埃希菌潜在致病风险

目的 对我国不同来源产志贺毒素大肠埃希菌（STEC）分离株的致病潜力进行分级,为STEC的风险管理提供参考依据。方法 运用联合国粮食及农业组织和世界卫生组织于2018年联合发布的STEC与食品归因、表征和监测报告中提出的危害等级分级方法,对我国2018—2022年已发表的STEC数据进行分级分析。结果 分级结果显示,纳入研究的STEC菌株中72.9%为低危害菌株,26.0%为高危害菌株,1.1%为最高危害菌株。高危害菌株主要来自牛或牛肉食品（95.3%）,仅有8株为最高危害菌株,分别来自牛和患者。结论 仅依据STEC菌株的血清型不足以作为菌株毒力评判标准,通过毒力基因组合对STEC感染风险进行分级是更可靠的方法。这一方法可为我国制定STEC监测和风险评估提供参考。     

黄竹生长过程中纤维形态与结构的演变过程研究

本研究以黄竹为实验原料,借助场发射扫描电子显微镜、二维广角X射线衍射仪等对其生长过程中竹纤维形态、结构的演变过程以及纤维物理性能进行了研究。结果表明,竹龄相同时,黄竹笋期基部的分化发育早于上部;随竹龄增大,前10月细胞壁增厚明显,竹龄3月时壁腔比为0.10,36月时为4.03;随着细胞壁逐渐增厚,纤维由柔软、易弯曲变得硬挺。通过场发射扫描电子显微镜观察,纤维由扁平状逐渐变为多棱柱或近似圆柱体;黄竹纤维的纤维平均长度、平均宽度增大;同一竹龄黄竹纤维的微纤丝角从近竹青至近竹黄逐渐增大,随黄竹径向高度的增大呈现波动趋势,整体在9°~14°范围内,纤维取向良好。     

Effect of modified atmosphere and temperature abuse on the growth from spores and cereulide production of Bacillus weihenstephanensis in a cooked chilled meat sausage

The effect of modified atmosphere packaging (MAP) on the germination and growth of toxin producing psychrotolerant Bacillus spp is not well described. A model agar system mimicking a cooked meat product was used in initial experiments. Incubation at refrigeration temperature of 8 °C for 5 weeks of 26 Bacillus weihenstephanensis including two emetic toxin (cereulide) producing strains showed that B. weihenstephanensis is sensitive to MAP containing CO₂. The sensitivity to 20% CO₂ was dependent on strain and oxygen level, being increased when oxygen was excluded from the MAP. Growth from spores was observed at the earliest within 2 weeks when 20% CO₂ was combined with 2% O₂ and in 3 weeks when combined with “0”% O₂ (the remaining atmosphere was made up from N₂). Results were validated in a cooked meat sausage model for two non-emetic and one emetic B. weihenstephanensis strain. The packaging film oxygen transfer rates (OTR) were 1.3 and 40 ml/m²/24 h and the atmospheres were 2% O₂/20% CO₂ and “0”% O₂/20% CO₂. Oxygen availability had a large impact on the growth from spores in the MAP meat sausage, only the most oxygen restricted condition (OTR of 1.3 ml/m²/24 h and “0”% O₂/20 % CO₂) inhibited growth of the three strains during 4 weeks storage at 8 °C. Cereulide production was undetectable during storage at 8 °C irrespective of choice of the MAP (quantified by liquid chromatography mass spectrometry/mass spectrometry). MAP storage at 8 °C for 1 and 3 weeks followed by opening of packages and temperature abuse for 1.5 h daily at 20 °C during 1 week resulted in increased cell counts and variable cereulide production in the meat sausage. A pre-history at 8 °C for 1 week in MAP with OTR of 1.3 or 40 ml/m²/24 h and 2% O₂ resulted in cereulide concentrations of 0.816 – 1.353 µg/g meat sausage, while a pre-history under the most oxygen restricted condition (OTR of 1.3 ml/m²/24 h, “0”% O₂/20 % CO₂) resulted in minimal cereulide production (0.004 µg/g meat sausage) at abuse condition. Extension of MAP storage at 8 °C for 3 weeks followed by abuse resulted in a substantially reduced cereulide production.Data demonstrates that MAP can be used to inhibit growth of a psychrotolerant toxin producing Bacillus spp. during chill storage at 8 °C, and substantially reduce the risk of emetic food poisoning at abuse condition. Results are of relevance for improving safety of ready to eat processed chilled foods of extended durability.     

Improvement and induction property of radiation-responsive promoter through DNA shuffling of 5′-flanking regions of the human p21 gene

A promoter that augments gene expression in response to stimulation of ionizing radiation would be a desired tool for radiogenetic therapy, a combination of radiotherapy and gene therapy. Although various promoters occurring naturally or artificially have been used for researches, one showing higher reactivity to ionizing radiation is desirable. In the present study, we attempted to improve a radiation-responsive promoter of the p21 through a technique called DNA shuffling. A library of DNA fragments was constructed by re-ligation of randomly digested promoter fragments and improved promoters were chosen out of the library. We repeated this process twice to obtain a promoter showing 2.6-fold better reactivity to ionizing radiation compared with its parent, p21 promoter after 10 Gy γ-ray irradiation. Nucleotide sequence analyses revealed that the obtained promoter was densely packed with some of the cis-acting elements including binding sites for p53, NF-κB, NRF-2, AP-1 and NF-Y more than p21 promoter. In addition, it was shown that its induction by ionizing radiation was dependent upon p53 status of a cell line, suggesting that the promoter retained properties of the p21 promoter. This technique is simple and efficient to improve a promoter responsive to other stimulus of interest besides IR.     

Cloning and expression of the gene encoding the thermophilic NAD(P)H-FMN oxidoreductase coupling with the desulfurization enzymes from Paenibacillus sp. A11-2

The gene encoding the NAD(P)H-flavin oxidoreductase (flavin reductase) which couples with the thermophilic dibenzothiophene (DBT)-desulfurizing monooxygenases of Paenibacillus sp. A11-2 was cloned in Escherichia coli and designated tdsD. Nucleotide sequence analysis suggested that the gene product consisted of 200 amino acids and showed about 30%, 27% and 26% amino acid sequence similarity to the major flavin reductase of Vibrio fischeri, the NADH dehydrogenase of Thermus thermophilus and several oxygen-insensitive NAD(P)H nitroreductases in the Enterobacteriaceae family, respectively. Both the growing and resting recombinant E. coli, in which tdsD was coexpressed with a set of desulfurizing genes, showed a rate of DBT removal about 5 times higher than the recombinants lacking tdsD. Maximal desulfurization was observed close to 45°C and 55°C in the resting cells and in the cell-free extraction reaction with the tdsD-coexpressing recombinants, respectively. In an organic/aqueous biphasic system, the coexpression of tdsD also markedly enhanced the rate of DBT removal.     

2017—2020年江苏省无锡市沙门菌的血清型与耐药性研究

目的 分析2017—2020年江苏省无锡市沙门菌报告病例的血清型特点和抗生素耐药性流行特征及变化趋势。方法 收集分离自 2017—2020年无锡市食源性疾病哨点监测医院腹泻病例（住院和门诊患者）标本中的216株沙门菌菌株,采用玻片凝集法对216株菌进行血清型鉴定及分析,并采用微量肉汤稀释法检测沙门菌对13种抗生素的药物敏感性。结果 216株沙门菌分为42种血清型,优势血清型分别为肠炎沙门菌28.70%（62/216）占比,鼠伤寒沙门菌26.39%（57/216）。耐药性分析结果显示,216株沙门菌对美洛培南、阿米卡星、阿奇霉素和头孢他啶高度敏感,高敏菌株占比均高于90%;所有沙门菌对氨苄西林的耐药率最高,达到68.06%,对美洛培南的耐药率最低,仅0.46%;沙门菌在2017—2020年对氨苄西林的耐药率呈逐年增高的趋势,且每年沙门菌分离株对氨苄西林耐药性最高;共130株菌株产生了多重耐药性（60.19%）,耐药菌株数最多的耐药谱为AMP-TET-STR,占比5.56%（12/216）,优势耐药谱不明显。结论 无锡市沙门菌的流行血清型以肠炎沙门菌和鼠伤寒沙门菌为主,且沙门菌耐药性形势严峻,应该尽快建立高效的管控机制, 加强药敏监测, 优化治疗方案, 避免抗生素滥用。     

Simulation of the effect of sausage ingredients and technology on the functionality of the bacteriocin-producing Lactobacillus sakei CTC 494 strain

Bacteriocin-producing lactic acid bacteria may be applied as novel functional starter cultures for sausage fermentation. In this way, safer and more standardised end products may be obtained. However, it is not clear how such strains behave under sausage fermentation conditions. In this study, the combined effects of typical sausage ingredients and process technology on the functionality of Lactobacillus sakei CTC 494 were simulated by a modelling approach. Under simulated sausage fermentation conditions, the strain was able to produce a considerable amount of bacteriocin. Model simulations indicate that sausage fermentation conditions of temperature and pH favour bacteriocin production, whereas salting and curing with sodium nitrite decrease growth and bacteriocin production. Sodium nitrite inhibits cell growth under its undissociated nitrous acid form, and its inhibitory effect seems to parallel lactic acid production. Whereas oxygen and magnesium levels did not influence bacterial functionality, manganese limitations severely decreased cell growth. Moreover, the presence of large amounts of fat, which is typical for a sausage environment, leads to an apparent bacteriocin inactivation, probably due to adsorption of the bacteriocin molecules from the water phase to the fat particles.     

Phylogenetic analysis of Rhodococcus erythropolis based on the variation of ribosomal proteins as observed by matrix-assisted laser desorption ionization-mass spectrometry without using genome information

Rhodococcus erythropolis strains characterized as antibiotic producers can be classified into three groups according to their antibiotic spectrum and growth compatibility. Due to their high genotypic similarity, the taxonomic relationship of these strains has not been elucidated. In this study, ribosomal protein profiling using matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS) was employed to classify twenty-one strains of R. erythropolis (15 antibiotic producers and 6 non-antibiotic producers). In the first step in this method, a total of 30 intense peaks observed for purified ribosomal subunit proteins of the type strain (R. erythropolis JCM 3201^T) were selected as the reference peaks. The mass spectra observed for the cell lysates of each sample strain were then checked as to whether peaks were observed at the same masses of the reference peaks. The results of peak matching were processed by cluster analysis, generating a dendrogram. Four major clusters of the R. erythropolis strains corresponded to three antibiotic groups and the non-antibiotic group. Furthermore, the topology of the dendrogram was highly comparable with the phylogenetic tree based on DNA gyrase subunit B gene (gyrB) sequencing. These results indicate that our proposed ribosomal protein profiling method using MALDI-MS is a potentially reliable and sufficiently high-throughput technique for the taxonomic analysis of closely related bacterial strains without using DNA sequence information.