Protein Containing the GGDEF Domain Affects Motility and Biofilm Formation in Vibrio cholerae and is Negatively Regulated by Fur and HapR

Objective This study aimed to investigate whether the VCA0560 gene acts as an active diguanylate cyclase(DGC)in Vibrio cholerae and how its transcription is regulated by Fur and Hap R.Methods The roles of VCA0560 was investigated by utilizing various phenotypic assays,including colony morphological characterization,crystal violet staining,Cyclic di-GMP(c-di-GMP)quantification,and swimming motility assay.The regulation of the VCA0560 gene by Fur and Hap R was analyzed by luminescence assay,electrophoretic mobility shift assay,and DNase I footprinting.Results VCA0560 gene mutation did not affect biofilm formation,motility,and c-di-GMP synthesis in V.cholerae,and its overexpression remarkably enhanced biofilm formation and intracellular c-di-GMP level but reduced motility capacity.The transcription of the VCA0560 gene was directly repressed by Fur and the master quorum sensing regulator Hap R.Conclusion Overexpressed VCA0560 functions as an active DGC in V.cholerae,and its transcription is repressed by Fur and Hap R.

Acquisition and dissemination mechanisms of CTXΦ in Vibrio cholerae : New paradigm for dif residents

Vibrio cholerae(V. cholerae) genome is equipped with a number of integrative mobile genetic element(IMGE) like prophages, plasmids, transposons or genomic islands, which provides fitness factors that help the pathogen to survive in changing environmental conditions. Metagenomic analyses of clinical and environmental V. cholerae isolates revealed that dimer resolution sites(dif) harbor several structurally and functionally distinct IMGEs. All IMGEs present in the dif region exploit chromosomally encoded tyrosine recombinases, Xer C and Xer D, for integration. Integration takes place due to site-specific recombination between two specific DNA sequences; chromosomal sequence is called att B and IMGEs sequence is called att P. Different IMGEs present in the att P region have different attP structure but all of them are recognized by Xer C and Xer D enzymes and mediate either reversible or irreversible integration. Cholera toxin phage(CTXΦ), a lysogenic filamentous phage carrying the cholera toxin genes ctx AB, deserves special attention because it provides V. cholerae the crucial toxin and is always present in the dif region of all epidemic cholera isolates. Therefore, understanding the mechanisms of integration and dissemination of CTXΦ, genetic and ecological factors which support CTXΦ integration as well as production of virion from chromosomally integrated phage genome and interactions of CTXΦ with other genetic elements present in the genomes of V. cholerae is important for learning more about the biology of cholera pathogen.

Evaluation of Technical Education by Using a Modern Structured MOODLE Laboratory Course, in Relation to Recent Data

E-learning platforms support education systems worldwide, transferring theoretical knowledge as well as soft skills. In the present study high-school pupils’, and adult students’ opinions were evaluated through a modern structured MOODLE interactive course, designed for the needs of the laboratory course “Automotive Systems”. The study concerns Greek secondary vocational education pupils aged 18 and vocational training adult students aged 20 to 50 years. The multistage, equal size simple random cluster sample was used as a sampling method. Pupils and adult students of each cluster completed structured 10-question questionnaires both before and after attending the course. A total of 120 questionnaires were collected. In general, our findings disclosed that the majority of pupils and adult students had significantly improved their knowledge and skills from using MOODLE. They reported strengthening conventional teaching, using the new MOODLE technology. The satisfaction indices improved quite, with the differences in their mean values being statistically significant.

Plasticity of Regulation of Mannitol Phosphotransferase System Operon by CRP-cAMP Complex in Vibrio cholerae

Objective The complex of the cyclic AMP receptor protein （CRP） and cAMP is an important transcriptional regulator of numerous genes in prokaryotes. The transport of mannitol through the phosphotransferase systems （PTS） is regulated by the CRP-cAMP complex. The aim of the study is to investigate how the CRP-cAMP complex acting on the mannitol PTS operon mtl of the Vibrio cholerae El Tot biotype. Methods The crp mutant strain was generated by homologous recombination to assess the need of CRP to activate the mannitol PTS operon of V. choleroe El Tor. Electrophoretic mobility shift assays （EMSA） and the reporter plasmid pBBRlux were used to confirm the role that the CRP-cAMP complex playing on the mannitol PTS operon intl. Results In this study, we confirmed that CRP is strictly needed for the activation of the mtl operon. We further experimentally identified five CRP binding sites within the promoter region upstream of the mannitol PTS operon mtl of the Vibrio cholerae El Tor biotype and found that these sites display different affinities for CRP and provide different contributions to the activation of the operon. Conclusion The five binding sites collectively confer the strong activation of mannitol transfer by CRP in V. choleroe, indicating an elaborate and subtle CRP activation mechanism.

Structural Variation of the Superintegron in the Toxigenic Vibrio cholerae O1 El Tor

Objective To understand the genetic structures and variations of the superintegron （Sl） in Vibrio cholerae isolated in the seventh cholera pandemic. Methods Polymerase chain reaction scanning and fragment sequencing were used. Sixty toxigenic V. cholerae O1 El Tor strains isolated between 1961 and 2008 were analyzed. Results Some variations were found, including insertions, replacements, and deletions. Most of the deletions were probably the result of recombination between V. cholerae repeat sequences. The majority of the variations clustered together. The Sis of the strains isolated in the 1960s and 1970s showed more diversity, whereas SI cassette variations in strains isolated in the 1990s and after were lower, with -24 kb signature sequence deletion. This indicates the predominant Sl in the host during the epidemic in the 1990s and after. The insertion cassettes suggested the mobilization from the Sls of other V. cholerae serogroups and Vibrio mimicus. Conclusion The study revealed that structural variations of Sis were obvious in the strains isolated in epidemics in different decades, whereas the divergence was based on syntenic structure of Sis in these El Tot strains. Also, the continuing cassette flows in the Sis of the host strains during the seventh cholera pandemics were displayed.

Specific Detection of Toxigenic Vibrio cholerae Based on in situ PCR in Combination With Flow Cytometry

Objective To develop an in situ PCR in combination with flow cytometry （ISPCR-FCM） for monitoring cholera toxin positive Vibrio cholerae. Methods In running this method, 4% paraformaldehyde was used to fix the Vibrio cholerae cells and 1 mg/mL lysozyme for 20 min to permeabilize the cells. Before the PCR thermal cycling, 2.5% glycerol was added into the PCR reaction mixture in order to protect the integrality of the cells. Results A length of 1037bp DNA sequence was amplified, which is specific for the cholera toxin gene （ctxAB gene）. Cells subjected to ISPCR showed the presences of ctxAB gene both in epifluorescence microscopy and in flow cytometric analysis. The specificity and sensitivity of the method were investigated. The sensitivity was relatively low （10^5 cells/mL）, while the specificity was high. Conclusion We have successfully developed a new technique for detection of toxigenic Vibrio cholerae strains. Further study is needed to enhance its sensitivities. ISPCR-FCM shows a great promise in monitoring specific bacteria and their physiological states in environmental samples.

Survival and proliferation of the lysogenic bacteriophage CTXΦ in Vibrio cholerae

The lysogenic phage CTXΦ of Vibrio cholerae can transfer the cholera toxin gene both horizontally(inter-strain) and vertically(cell proliferation). Due to its diversity in form and species, the complexity of regulatory mechanisms, and the important role of the infection mechanism in the production of new virulent strains of V.cholerae, the study of the lysogenic phage CTXΦ has attracted much attention. Based on the progress of current research, the genomic features and their arrangement, the host-dependent regulatory mechanisms of CTXΦ phage survival, proliferation and propagation were reviewed to further understand the phage's role in the evolutionary and epidemiological mechanisms of V. cholerae.

Occurrence of <i>Vibrio cholerae</i>in Some Households Engaged in Livestock Farming in Some Parts of Zaria, Nigeria

The study was designed to determine the presence of Vibrio cholerae from the environment in close proximity with livestock in Zaria,Kadunastate. Three hundred and thirty six environmental samples comprising soil, water, manure and vegetables were collected from some selected households inZariacoveringZariacity, Sabo and Samaru from April to October 2006 and analyzed for the presence of Vibrio cholerae, one of the etiologic agent of gastroenteritis. Twelve Vibrios exhibiting characteristics of Vibrio cholerae were identified using biochemical techniques. Serological identification confirmed 5 (41.7%) of these isolates as Vibrio, 1 (20%) as Vibrio serogroup O1, Ogawa biotype. A 0.59% prevalence was obtained for this pathogen in the study. The 0.59% isolation rate though low is significant considering it’s source being animal, since livestock keeping is a common feature in the study location, with animals living in close proximity to man this work is imperative. Animals are a point source of contamination of enteric pathogens, therefore extensive management system and proper treatment of animal manure is recommended before its use as fertilizer.

泥鳅(Misgurnus anguillicaudatus)病原霍乱弧菌(Vibrio cholerae)PCR与LAMP检测方法的比较研究

采用分子生物学方法,以霍乱弧菌lolB为靶基因设计特异性引物,进行了霍乱弧菌的PCR和环介导等温核酸扩增(Loop-mediatedisothermal amplification,LAMP)检测技术研究,并对它们的特异性、灵敏性和实际应用进行了比较。结果表明,所建立的PCR检测霍乱弧菌的方法最低检测限为4.0×103CFU/ml;LAMP检测方法在65℃下恒温扩增60min,检测限为4.0×101CFU/ml,反应产物加入荧光染料SYBRGreenI后反应液呈现明显的绿色;以温和气单胞菌、副溶血弧菌、鳗弧菌及美人鱼弧菌为对照菌株,检测结果均为阴性;霍乱弧菌人工染菌的8种水产品进行PCR及LAMP检测,结果均为阳性,而未染菌组均为阴性;PCR及LAMP检测霍乱弧菌的方法均具有灵敏度较高、特异性强等优点,且LAMP检测霍乱弧菌的方法灵敏度是PCR方法的100倍,更适合于养殖现场检测的推广使用。

泥鳅(Misgurnus anguillicaudatus)病原霍乱弧菌(Vibrio cholerae)的表型与分子鉴定

从江苏连云港某养殖场养殖死亡的泥鳅肝脏、血液及腹水中分离出大量优势生长的细菌,人工感染试验证明其对泥鳅具有很强的致病性。采用表观分类学及分子生物学方法,对分离菌进行了形态特征、理化特性、胞外酶活性及溶血活性等生物学性状检验;用PCR方法同时扩增其16SrRNA和gyrB基因,分析了16SrRNA和gyrB两种基因序列的同源性,并构建了系统发生树,通过基因序列分析,比较了两种基因在相似细菌的检测和鉴定能力;基于16SrRNA和gyrB基因的系统发育学分析表明分离菌(LD081008B-1)所扩增的16SrRNA和gyrB基因序列均与GenBank数据库中霍乱弧菌具有较高的相似性,且gyrB基因用于细菌种间鉴定更具优越性;16SrRNA基因序列长度为1446bp(GenBank登录号:GQ205447),gyrB基因序列长度为1207bp(GenBank登录号:GQ205452);根据分离菌的表型特征及分子特征,判定病原菌为弧菌属(VibrioPacini1854)的霍乱弧菌(Vibriocholerae)。胞外酶活性及溶血活性检测表明分离菌均具有蛋白酶、卵磷脂酶、淀粉酶、明胶酶及DNA酶活性,在含7%家兔脱纤血液营养琼脂培养基上呈β型溶血。分离菌的耐药谱测定结果显示,除对供试49种抗菌药物中的杆菌肽耐药外,对其它48种药物敏感或高度敏感。

Design of a multiplex PCR method for detection of toxigenicpathogenic in Vibrio cholerae

Objective:To study virulence and regulatory genes(hlyA,ctxB,tcpI) in clinical strains of Vibrio ckolerae(V.cholerae),simultaneously.Methods:Three important genes,tepI,hlyA and ctxB were used for detection of toxigenic and pathogenic V.cholera by chain reaction assay method. Results:According to the results of the PCR,the incidence of hlyA,tcpI,and ctxB genes in clinical isolates was obtained as 94.7%(72 sample),90.8%(69 sample),and 92.1%(70 sample), respectively.Five strains possessed all genes except ctxB,six strains possessed all genes except tcpI,four strains possessed all genes except hlyA,one strain possessed only hlyA and 60 strains contained a combination of three genes.Including hlyA,ctxB and tcpI,Conclusions:Result show that this method could be reliable to detect toxigenic-pathogenic strains of V.cholerae in Iran.

Comparative proteomic analysis of Vibrio cholerae O139, O22, O155 and El Tor biotype epidemic strains

The purposes of this study are to compare the proteome of Vibrio cholerae O139 strains with that of O22, O155 and El Tot biotype epidemic strains, and to identifv whether O139 strains have a close relation with the latter. Proteins of two V. cholerae serogroup O139 strains, two El Tor biotype epidemic strains, one O22 strain, and one O155 strain were separated by two-dimensional gel electrophoresis （2-DE） ; the silver stained 2-DE gels were scanned with （ligital lmageScanner and analyzed with lmageMaster 2D Elite 3.10 software. Similarities of protein maps between these strains were analyzed. The two O139 strains had 86% proteins in common; the two El Tor biotype epidemie strains showed 84% proteins in common; O139 strains shared 67 % , 47 % and 45 % proteins with El Tor biotype epidemic strains, O22 and O 155 strains respectively. Although the proteome of O 139 strains have close similarity to that of El Tor biotype epidemic strains, great disparities exist. O139 strains may not originate from El Tor biotype directly, and a transition strain may exist.

Plasmid mediated antibiotic resistance of Vibrio cholerae Ol biotype E1 Tor serotype Ogawa associated with an outbreak in Kolkata,India

Objective:To determine the antibiotic resistance of Vibrio choleras(V.cholerae) O1 biotype El Tor serotype Ogawa isolates involved in an outbreak of watery diarrhea in Kolkata,and to explore the role of plasmid in mediating antibiotic resistance.Methods:Antibiotic susceptibility and minimum inhibitory concentration(MIC) values of antibiotics for the isolated V.cholerae 01 Ogawa(n=12) were determined by disk diffusion and agar dilution methods,respectively,using ampicillin(Am),chloramphenicol(C),trimethoprim(Tm),tetracycline(T).erythromycine(Er), nalidixic acid(Nx).ciprofloxacin(Cp),amikacin(Ak) and cefotaxime(Cf).Plasmid curing of multidrug resistant(MDR) V.cholerae 01 Ogawa strains was done following ethidium bromide treatment.Following electrophoresis,the plasmid DNAs,extracted from the isolated MDR V. cholerae O1 Ogawa strains and their cured derivatives,were visualized and documented in ’gel doc’ system.Results:The outbreak causing V.cholerae O1 Ogawa isolates were MDR as determined by disk diffusion susceptibility test,and MIC determination.The isolates showed three different drug resistance patterns:AmTmTErNx(for 6 isolates).TmTErCp(for 5 isolates), and AmTniNx(for one isolate),and showed uniform sensitivity to C,Ak and Cf.The loss of plasmids with the concomitant loss of resistance to Am,Tm,T and Er of the isolates occurred following ethidium bromide treatment.Conclusions:The current findings suggest that the V. cholerae O1 Ogawa associated with the cholera outbreak were MDR,and resistance to Am,Tm,T and Er among the isolates were plasmid mediated.

霍乱弧菌(Vibrio cholerae)的细胞形态研究——活的非可培养状态细胞

使用透射电镜和萤光抗体染色，对处于可培养和活的非可培养状态的霍乱弧菌进行了细胞形态结构比较研究。结果表明：处于非可培养状态的霍乱菌细胞，体积明显地缩小，形态由弧形变成了球形，我们共观察了３８２个非可培养状态的细胞，发现它们的细胞壁都是完整的，即不是细菌－Ｌ型。

基于ⅢF A/V规范和Avalon系统的大学图书馆视听数据库建设研究

随着中国网络基础设施的不断改善,视听媒体在年轻一代中非常流行,给以文本资源为主的图书馆带来了挑战。本研究旨在探究国内外大学图书馆视听资源数据库建设的现状,借鉴ⅢF规范在图像资源管理方面的成功经验和各种视听保存社区的实践,提出基于ⅢF A/V规范与开源软件的中国大学图书馆视听资源管理方法。通过分析华东师范大学图书馆在视听资源保存、流媒体发布、时间轴气泡注释、转录、视听结构化和开放共享方面的实践,进行实证研究。

基于改进YOLO v7轻量化模型的自然果园环境下苹果识别方法

针对自然果园环境下苹果果实识别中,传统的目标检测算法往往很难在检测模型的检测精度、速度和轻量化方面实现平衡,提出了一种基于改进YOLO v7的轻量化苹果检测模型。首先,引入部分卷积(Partial convolution, PConv)替换多分支堆叠模块中的部分常规卷积进行轻量化改进,以降低模型的参数量和计算量;其次,添加轻量化的高效通道注意力(Efficient channel attention, ECA)模块以提高网络的特征提取能力,改善复杂环境下遮挡目标的错检漏检问题;在模型训练过程中采用基于麻雀搜索算法(Sparrow search algorithm, SSA)的学习率优化策略来进一步提高模型的检测精度。试验结果显示:相比于YOLO v7原始模型,改进后模型的精确率、召回率和平均精度分别提高4.15、0.38、1.39个百分点,其参数量和计算量分别降低22.93%和27.41%,在GPU和CPU上检测单幅图像的平均用时分别减少0.003 s和0.014 s。结果表明,改进后的模型可以实时准确地识别复杂果园环境中的苹果,模型参数量和计算量较小,适合部署于苹果采摘机器人的嵌入式设备上,为实现苹果的无人化智能采摘奠定了基础。

基于改进轻量化YOLO v5n的番茄叶片病害识别方法

针对现有番茄叶片病害识别存在背景复杂、识别准确率低、模型参数量大、计算量大以及难以部署至移动设备或嵌入式设备等问题,提出一种改进的轻量化YOLO v5n的番茄叶片病害识别方法。首先收集细菌性斑疹病、早疫病、晚疫病、叶霉病、斑枯病、褐斑病等6种常见番茄叶片病害图像以及番茄健康叶片图像,对图像进行镜像翻转、高斯模糊等数据增强方式增加样本多样性,提升模型识别和泛化能力。接着在YOLO v5n网络基础上,选择采用轻量化的C3Ghost模块替换C3模块以压缩卷积过程中的计算量、模型权重和大小,同时在颈部网络中融合轻量级卷积技术GSConv和VOV-GSCSP模块,在增强特征提取能力的同时降低模型参数量。最后引入PAGCP算法对改进后的模型进行全局通道剪枝压缩参数量并减少训练开销。试验结果表明,改进后的YOLO v5n平均精度均值达到99.0%,参数量减少66.67%,计算量降低了2.6 G,模型权重压缩了2.23 MB。本研究提出的番茄叶片病害识别方法在降低了模型大小、参数量、计算量的同时仍保持较高的识别精度,为移动设备上实现番茄叶片病害识别提供技术参考。

Isolation, Identification and Antimicrobial Susceptibility Profile Analysis of <i>Vibrio cholerae</i>O1 from Stool Samples of Bangladesh

Cholera is a severe diarrheal disease which is usually caused by toxigenic strain of Vibrio cholerae O1 and O139. Cholera is still one of the major health concerns in developing countries like Bangladesh due to poor sanitation and unavailability of safe drinking water. This experiment was confronted to identify V. cholerae O1 from stool samples as well as to determine the antibiotic susceptibility pattern of the isolated strains. A total of 140 stool samples from people infected with diarrheal disease were collected from July 2016 to December 2016. Among all, 58 samples were found positive for V. cholerae which were further subjected to sero-grouping by specific anti-sera and antimicrobial sus-ceptibility test by Kirby Bauer disc diffusion method. The zones of inhibition were measured and interpreted by following the recommendations of the criteria of Clinical and Laboratory Standards Institute (CLSI). It was found that 43 (74.1%) isolates of V. cholerae were O1 serogroup of Ogawa serotype and the rest 15 (25.9%) were O1 serogroup of Inaba serotype. People aged between 41 - 50 were most susceptible to V. cholerae O1 having about 39.7% of positive cases. The isolates were highly susceptible to Ciprofloxacin and Gentamicin with 100% susceptibility whereas 100% resistant was found towards Nalidixic acid. Though most of the isolates in our study were susceptible against tested antibiotics, the continuous surveillance is required to see the changing pattern of serogroups or serotypes and antimicrobial profile in this region.

Geospatial Variability of Cholera Cases in Malawi Based on Climatic and Socioeconomic Influences

Cholera remains a public health threat in most developing countries in Asia and Africa including Malawi with seasonal recurrent outbreaks. Malawi’s recent Cholera outbreak in 2022 and 2023, exhibited higher morbidity and mortality rates than the past two decades. Lack of spatiotemporal-based technology and variability assessment tools in Malawi’s Cholera monitoring and management, limit our understanding of the disease’s epidemiology. The present work developed a spatiotemporal variability model for Cholera disease at district level and its relationship to socioeconomic and climatic factors based on cumulative confirmed Cholera cases in Malawi from March 2022 to July 2023 using Z-score statistic and multiscale geographically weighted regression (MGWR) in a Geographical Information System (GIS). We found out that socioeconomic factors such as access to safe drinking water, population density and poverty level, and climatic factors including temperature and rainfall strongly influenced Cholera prevalence in a complex and multifaceted manner. The model shows that Lilongwe, Mangochi, Blantyre and Balaka districts were highly vulnerable to Cholera disease followed by lakeshore districts of Salima, Nkhotakota, Nkhata-Bay and Karonga than other districts. We recommend strategic measures such as Water, Sanitation, and Hygiene (WASH) interventions, community awareness on proper water storage, Cholera case management, vaccination campaigns and spatial-based surveillance systems in the most affected districts. This research has shown that MGWR, as a surveillance system, has the potential of providing insights on the disease’s spatial patterns for public health authorities to identify high-risk districts and implement early response interventions to reduce the spread of the disease.

基于改进DeepLabv3+的轻量化作物杂草识别方法

为在存储资源与计算能力有限的设备上实现田间作物和杂草的识别,本文提出一种基于改进DeepLabv3+的轻量化语义分割网络。首先,以MobileNet v2作为DeepLabv3+的特征提取骨干网络,提出双分支残差模块替换倒残差模块,并删除后两层卷积以降低模型参数量。其次,在空洞空间金字塔池化(Atrous Spatial Pyramid Pooling,ASPP)模块中引入分组逐点卷积,使用深度扩张卷积替换标准卷积,并将卷积后的特征图进行多尺度特征融合增强对作物和杂草深层特征的提取能力。最后,将原有的非线性激活函数替换为Leaky ReLU激活函数来提升分割精度。实验结果表明:改进后网络的mIOU达到86.75%,参数量仅为0.69M,FPS达到了98,与原始DeepLabv3+以及3个典型轻量化语义分割网络的相比,参数量最小,在对比的轻量化网络中具有最高的分割精度。