海桑属植物分子鉴定的ISSR分析

为准确鉴别海桑属植物,采用ISSR（inter-simple sequence repeat）分子标记技术,对6种海桑属植物(海桑、拟海桑、杯萼海桑、海南海桑、卵叶海桑及无瓣海桑)基因组DNA进行PCR扩增。建立了海桑属植物ISSR标记的标准程序和海桑属各物种的DNA指纹数据库。采用的11个引物共产生71个物种特征性标记,其中无瓣海桑23个,海南海桑16个,海桑15个,杯萼海桑6个,拟海桑6个,卵叶海桑5个。运用这些特征性标记,可迅速区分海桑属植物。研究表明ISSR技术是在分子水平上鉴定海桑属植物的一种行之有效的方法。     

Hemolytic Factor Production by Clinical Isolates of Candida Species

Most cases of fungal bloodstream infections (BIs) are attributed to Candida albicans; however, non-Candida albicans Candida species have recently been identified as common pathogens. Although hemolytic factor is known to be putative virulence factor contributing to pathogenicity in Candida species, its production is poorly evaluated. The present study was undertaken to analyze the production of hemolytic factor by C. albicans (10), C. tropicalis (13), and C. parapsilosis (8) isolates associated with BIs. Data of hemolysis zones on plate assay revealed that the majority of C. albicans isolates produced mild hemolytic activity whereas the majority of C. tropicalis produced strong activity. None of the tested C. parapsilosis isolates exhibited hemolysis on plate assay. We also evaluated the hemolytic activity in the cell-free broth. There were no significant differences (P > 0.05) in the secreted hemolytic activity among intra-species isolates. Different levels of secreted hemolytic factor were observed for Candida species, where C. tropicalis exhibited the highest production of hemolytic factor (P < 0.05) followed by C. albicans and C. parapsilosis. Inhibition of hemolysis (up to 89.12 %) from culture supernatant, following incubation with the lectin Concanavalin A (Con A), was observed for all three Candida species. This finding suggests that the secreted hemolytic factor of C. tropicalis and C. parapsilosis may be a mannoprotein, similar to that described for C. albicans.     

Comparison of Three Plant Rhabdovirus Isolates by Two Different Serological Techniques

Three different rhabdovirus isolates, eggplant mottled dwarf virus (EMDV), tomato veinyellowing virus (TVYV) and a hitherto undefined isolate from tomato in Portugal (Tom-P) were compared by two different serological techniques, immunosorbent electron microscopy (ISEM) and electro-blot-immunoassay (EBIA). Antisera were prepared either against purified virus or against partially purified virus preparations extracted by a one-step procedure. Even the partially purified virus preparations yielded antisera that allowed unequivocal identification of two of the five structural virus proteins, G and N, in EBIA and were suitable for specific trapping of complete virus particles on electron microscope grids. With both serological techniques identical results were obtained indicating a close serological relationship between the three isolates tested. Cross-reactions between the G proteins could be deduced from heterologous trapping of complete virus particles in ISEM. The cross-reactions for the G proteins were substantiated by the EBIA-results which indicated in addition cross-reactions between the remaining three major structural virus proteins, N, M₁, and M₂, but also revealed significant differences in the molecular weights of the G and M₁ proteins between TVYV and the other two isolates. The results obtained indicate that Tom-P is serologically closely related and in respect of protein molecular weights identical to EMDV, and that TVYV is not a separate virus but rather a strain of EMDV.     

Rapid Identification of Clinical Yeast Isolates by Mass Spectrometry

Historically, yeasts and molds have been identified by their microscopy and macroscopy appearance, detection of specific antigens and metabolites, the results of biochemical testing, and the presence of species-specific gene sequences. These methods are either nonspecific or, in the case of gene sequencing, technically demanding, time-consuming, and expensive. Recent studies have demonstrated that matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS) is a rapid, simple, inexpensive, and accurate tool for the identification of a wide spectrum of bacteria and fungi. There are also promising reports that this technology can be used for subtyping organisms and for performing antifungal susceptibility tests. This article reviews the applications of MALDI-TOF MS for routine clinical microbiology testing.     

Antimicrobial Combinations against Pan-Resistant Acinetobacter baumannii Isolates with Different Resistance Mechanisms

The study investigated the effect of antibiotic combinations against 20 clinical isolates of A. baumannii (seven colistin-resistant and 13 colistin-susceptible) with different resistance mechanisms. Clinical data, treatment, and patient mortality were evaluated. The following methods were used: MIC, PCRs, and outer membrane protein (OMP) analysis. Synergy was investigated using the checkerboard and time-kill methods. Clonality was evaluated by PFGE. Based on clonality, the whole genome sequence of six A. baumannii isolates was analyzed. All isolates were resistant to meropenem, rifampicin, and fosfomycin. OXA-23 and OXA-143 were the most frequent carbapenemases found. Four isolates showed loss of a 43kDa OMP_. The colistin-susceptible isolates belonged to different clones and showed the highest synergistic effect with fosfomycin-amikacin. Among colistin-resistant isolates, the highest synergistic effect was observed with the combinations of colistin-rifampicin followed by colistin-vancomycin. All colistin-resistant isolates harbored bla_OXA-23-like and belonged to CC113. Clinical and demographic data were available for 18 of 20 patients. Fourteen received treatment and eight patients died during treatment. The most frequent site of infection was the blood in 13 of 14 patients. Seven patients received vancomycin plus an active drug against A. baumannii; however, mortality did not differ in this group. The synergistic effect was similar for colistin-susceptible isolates of distinct clonal origin presenting with the same resistance mechanism. Overall mortality and death during treatment was high, and despite the high synergism in vitro with vancomycin, death did not differ comparing the use or not of vancomycin plus an active drug against A. baumannii.     

Lack of Genic Similarity between Two Sibling Species of Drosophila as Revealed by Varied Techniques

Acrylamide gel electrophoresis was performed on the enzyme xanthine dehydrogenase in sixty isochromosomal lines of Drosophila persimilis from three geographic populations. Sequential electrophoretic analysis using varied gel concentrations and buffers revealed twenty-three alleles in this species where only five had been described previously. These new electrophoretic techniques also detected a profound increase in divergence of gene frequencies at this locus between D. persimilis and its sibling species D. pseudoobscura. The implications of these results for questions of speciation and the maintenance of genetic variability are discussed.     

Transmission Comparisons of Cucumber Mosaic Virus Subgroup I and II Isolates by Different Aphid Species

To investigate the transmission differences between Cucumber mosaic virus (CMV) subgroup isolates, we carried out a comparative study with five aphid species Myzus persicae, Aphis gossypii, Lipaphis erysimi, Aphis craccivora and Megoura viciae in laboratory and field experiments to evaluate spread of CMV Subgroup I NX and subgroup II AG isolates in tobacco. Both NX and AG varied in transmission efficiency by the five aphids, and our transmission results revealed important differences in transmission efficiency of two isolates by Myzus persicae and Aphis gossypii. In contrast, significant transmission differences were not detected with Lipaphis erysimi, Aphis craccivora or Megoura viciae. Interestingly, the overall transmission efficiencies of the two different subgroup strains were almost equal when field transmissions were tested with mixed populations of the five aphid species. Our results together with our previously reported experiments on competition of CMV subgroup isolates in tobacco suggest that variations in aphid vector populations contribute substantially to the epidemic potential of CMV subgroup isolates.     

鲍曼不动杆菌的临床分布特点及耐药性分析

调查鲍曼不动杆菌的临床分布及其对抗菌药物的耐药情况,为临床合理用药提供依据。将哈尔滨医科大学第一附属医院临床各种来源的鲍曼不动杆菌1582株采用K-B法进行药敏试验,并对结果进行统计分析。2008至2010年共检出鲍曼不动杆菌1582株,临床分布以ICU最多(484株,占54.5%)。对抗菌药物的耐药率逐年增高,ICU抗菌药物的耐药率明显高于非ICU病区。该菌株对临床常用抗菌药物高度耐药和多重耐药,对亚胺培南和美罗培南耐药率高达90.9%和90.3%。鲍曼不动杆菌耐药情况相对严重,临床须重视鲍曼不动杆菌的感染,加强院內感染的控制及耐药性的监测,根据药敏结果选择合适抗生素,延缓耐药性进程。     

Diversity of Superoxide-Dismutases Among Clinical and Soil Isolates of Streptomyces Species

Comparison of the nature, activity, and cellular localization of superoxide-dismutases (SOD) from soil and clinical isolates of Streptomyces species was investigated to identify possible factors that could account for the pathological role of the strains isolated from human lesions. Results showed that all of the studied strains possessed a cytoplasmic Ni-SOD. This particular SOD, found in isolates from patients, could be a new taxonomic criterion to identify Streptomyces species with greater precision. A second minor SOD, assimilated to an Fe/Zn-SOD, was detected in some strains, but no relationship was established between the presence of this enzyme and the clinical origin of the strains. Received: 9 April 1999 / Accepted: 9 June 1999     

红雪茶基源种的鉴定及资源分布

本研究通过对红雪茶基源种进行考证,并结合品种鉴定及野外生态调查,结果表明,红雪茶的基源种应为金丝刷（Lethariella cladonioides）和金丝带（L.zahlbruckneri）,在这两个种群内,存在化学多态现象。市售的“红雪茶”主要由这两个种的四个化学宗所组成。红雪茶的分布中心主要在藏东南,滇西北及川西地区,目前红雪茶的资源已经受到了极大的破坏,日渐濒危。为加强对野生红雪茶资源的有效保护,提出了开展红雪茶种质资源保护的建议。     

临床分离肠球菌的分布特征及耐药性分析

目的:了解我院临床分离肠球菌的分布特征及耐药现状,为临床合理用药提供依据。方法:对我院2010年1月至2012年12月期间所有临床分离的肠球菌分布情况及药敏结果进行回顾性分析。结果:临床共分离肠球菌242株,粪肠球菌分离率(55.0%)高于屎肠球菌(40.9%),屎肠球菌分离率有增高的趋势。标本来源以尿液(62.9%)、分泌物(10.3%)、血液(6.9%)为主。肠球菌对万古霉素、替考拉宁的敏感性最高,均高于90%。发现耐万古霉素的肠球菌(VRE)7株,其中5株同时耐高浓度的氨基糖苷类抗生素(HLAR);对克林霉素、复方磺胺、阿米卡星、庆大霉素、妥布霉素、苯唑西林耐、头孢西丁耐药率最高,均高于95%。屎肠球菌对青霉素类、氨苄西林、红霉素、呋喃妥因、环丙沙星耐药率均高于粪肠球菌;对四环素、奎努普丁/达福普汀耐药率低于粪肠球菌。结论:肠球菌是临床感染重要病原菌,且具有多重耐药性,屎肠球菌和粪肠球菌耐药水平差异较大,临床应根据药敏结果合理选择抗菌药物。     

Original Species Identification and Geographic Distribution of Hongxuecha

The textual criticism of original species of Hongxuecha has been made combining species identification and field surveys. The results showed that Hongxuecha had two original species, Lethariella cladonioides and L.zahlbruckneri. There were chemical variability within the two populations. Hongxuecha in the market sales included four chemical races of the two species. The distribution center of Hongxuecha was mainly in the southeastern Tibet, western Sichuan and northwestern Yunnan. Currently, Hongxuecha resources have been greatly damaged and species are facing extinction. To enhance effective protection for wild hongxuecha resources, some proposals were presented.     

Distribution of Serratia Species in Clinical Specimens

Eighty-five strains of Serratia were speciated. The majority were Serratia marcescens and were recovered from a variety of clinical specimens. S. liquefaciens and S. rubidaea were recovered only from sputum.     

Identification and Pathogenic Potential of Clinical Bacillus and Paenibacillus Isolates

The soil-related Bacillus and Paenibacillus species have increasingly been implicated in various human diseases. Nevertheless, their identification still poses problems in the clinical microbiology laboratory and, with the exception of Bacillus anthracis and Bacillus cereus, little is known on their pathogenicity for humans. In this study, we evaluated the use of matrix-assisted laser desorption—ionization time of flight mass spectrometry (MALDI-TOF MS) in the identification of clinical isolates of these genera and conducted genotypic and phenotypic analyses to highlight specific virulence properties. Seventy-five clinical isolates were subjected to biochemical and MALDI-TOF MS identification. 16S rDNA sequencing and supplemental tests were used to solve any discrepancies or failures in the identification results. MALDI-TOF MS significantly outperformed classical biochemical testing for correct species identification and no misidentification was obtained. One third of the collected strains belonged to the B. cereus species, but also Bacillus pumilus and Bacillus subtilis were isolated at high rate. Antimicrobial susceptibility testing showed that all the B. cereus, B. licheniformis, B. simplex, B. mycoides, Paenibacillus glucanolyticus and Paenibacillus lautus isolates are resistant to penicillin. The evaluation of toxin/enzyme secretion, toxin-encoding genes, motility, and biofilm formation revealed that B. cereus displays the highest virulence potential. However, although generally considered nonpathogenic, most of the other species were shown to swim, swarm, produce biofilms, and secrete proteases that can have a role in bacterial virulence. In conclusion, MALDI-TOF MS appears useful for fast and accurate identification of Bacillus and Paenibacillus strains whose virulence properties make them of increasing clinical relevance.     

Protein Motional Details Revealed by Complementary Structural Biology Techniques

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利用ISSR揭示不同类型月季遗传多样性

本研究应用正交设计法对ISSR反应体系中的各个主要影响因子进行了优化筛选,确立了适合月季ISSR-PCR反应的最佳体系.结果表明,25μL的ISSR反应体系中各组分的最适浓度分别为:1×PCR缓冲液、1 U Taq DNA聚合酶、800 pmol/L引物、0.16 mmol/L dNTPs、Mg2+ 1.5 mmol/L.筛选了33个ISSR引物,共得到了11个多态性比较高的ISSR引物,占所筛引物的33.33%.利用筛选出的11条ISSR引物对3种月季类型的23份月季材料进行遗传多样性分析,共扩增出477条DNA带,其中多态性位点有14个,平均每条引物可以检测到4.5个多态性位点.用NTSYS软件对样品进行了UPGMA聚类分析,聚类结果表明丰花月季基本能聚为一类,切花月季与藤本月季交叉聚在一起.这表明月季种质的遗传差异与其应用分类的相关性不紧密.     

Distribution of Virulence Factors and Molecular Fingerprinting of Aeromonas Species Isolates from Water and Clinical Samples: Suggestive Evidence of Water-to-Human Transmission

A total of 227 isolates of Aeromonas obtained from different geographical locations in the United States and different parts of the world, including 28 reference strains, were analyzed to determine the presence of various virulence factors. These isolates were also fingerprinted using biochemical identification and pulse-field gel electrophoresis (PFGE). Of these 227 isolates, 199 that were collected from water and clinical samples belonged to three major groups or complexes, namely, the A. hydrophila group, the A. caviae-A. media group, and the A. veronii-A. sobria group, based on biochemical profiles, and they had various pulsotypes. When virulence factor activities were examined, Aeromonas isolates obtained from clinical sources had higher cytotoxic activities than isolates obtained from water sources for all three Aeromonas species groups. Likewise, the production of quorum-sensing signaling molecules, such as N-acyl homoserine lactone, was greater in clinical isolates than in isolates from water for the A. caviae-A. media and A. hydrophila groups. Based on colony blot DNA hybridization, the heat-labile cytotonic enterotoxin gene and the DNA adenosine methyltransferase gene were more prevalent in clinical isolates than in water isolates for all three Aeromonas groups. Using colony blot DNA hybridization and PFGE, we obtained three sets of water and clinical isolates that had the same virulence signature and had indistinguishable PFGE patterns. In addition, all of these isolates belonged to the A. caviae-A. media group. The findings of the present study provide the first suggestive evidence of successful colonization and infection by particular strains of certain Aeromonas species after transmission from water to humans.Aeromonas species cause both intestinal and extraintestinal infections (25, 33, 78), and the latter include septicemia, cellulitis, wound infections, urinary tract infections, hepatobiliary tract infections, soft tissue infections, and, occasionally, meningitis and peritonitis (25, 30, 78). In immunocompromised children, these pathogens can cause even more severe forms of infections, such as hemolytic-uremic syndrome (HUS) and necrotizing fasciitis (3, 23), although detailed studies are needed to establish such associations. Worldwide, the rate of isolation of Aeromonas from diarrheic stools has been reported to be as high as 10.8%, compared to 2.1% for healthy controls (25, 37, 78). An increased rate of isolation of Aeromonas species was reported in flood water samples during Hurricane Katrina in New Orleans (58), and skin and soft tissue infections caused by Aeromonas species were among the most common infections in the survivors of the 2004 tsunami in southern Thailand (28). In particular, Aeromonas salmonicida causes fish infections that result in huge economical losses in the fishing industry (6, 22). The ability of aeromonads, as well as other bacteria, to survive in chlorinated water when they are in biofilms and their resistance to multiple antibiotics are major public health concerns (46).Aeromonas-related gastroenteritis remains somewhat controversial (24, 36). There have been a number of well-described cases and a few documented outbreaks, but whether all aeromonad fecal isolates from symptomatic persons are the actual causes of diarrheal disease is still questionable. One theory for this conundrum was posed in 2000 by two of us, who suggested that only specific subsets of Aeromonas strains within and between species are actually pathogenic for humans (38). This highlights the importance of developing accurate biotyping, molecular fingerprinting, and virulence factor analysis methods for differentiating environmental and clinical aeromonads from one another and for comparing them (38).Of the 19 currently recognized Aeromonas species, A. hydrophila, A. caviae, and A. veronii biovar sobria are the most common species known to cause the majority of human infections, and they account for more than 85% of all clinical isolates (34). The pathogenesis of Aeromonas infections is multifactorial, as aeromonads produce a wide variety of virulence factors, including hemolysins, cytotonic and cytotoxic enterotoxins, proteases, lipases, leucocidins, endotoxin, adhesions, and an S layer, that act in concert to cause disease in the host (12-14, 50, 51). The cytotoxic enterotoxin Act, which has some similarities to aerolysin (31), is one of the most significant virulence factors in diarrheal isolate SSU of A. hydrophila and was first characterized in our laboratory (12). Act is secreted by the type II secretion system (T2SS) and has hemolytic, cytotoxic, and enterotoxic activities (12). In addition, our laboratory recently sequenced and characterized two other secretion systems, T3SS and T6SS, that were found to contribute to the virulence of A. hydrophila SSU (66, 67, 72). We recently characterized an effector of the T3SS, which was designated AexU, and found that it was associated with ADP ribosylation of host cell proteins, a rounded phenotype in HeLa cells, inhibition of phagocytosis, induction of apoptosis, and mouse mortality (66, 67). In recent studies, we also investigated the role of two T6SS-associated effectors, the valine-glycine repeat G (VgrG) family of proteins and hemolysin-coregulated protein (Hcp), in the virulence of A. hydrophila (71, 72). We demonstrated that VgrG1 of A. hydrophila had actin-ADP ribosylation activity that induced host cell cytotoxicity (71). Based on the model for T6SS, the VgrG1 protein must assemble with the highly homologous VgrG2 and VgrG3 proteins to form a cell-puncturing device to deliver effector proteins into the host cells (59). We also obtained evidence that expression of the hcp gene in HeLa cells led to their apoptosis, and animals immunized with recombinant Hcp were protected from subsequent challenge with a lethal dose of wild-type A. hydrophila SSU (72).In addition, cytotonic enterotoxins (e.g., Alt [heat labile] and Ast [heat stable]) were identified in a diarrheal A. hydrophila SSU isolate (14, 63) that induced fluid secretion in the ligated small intestinal loops of animals (47). More recently, we identified some additional virulence factor-encoding and regulatory genes, such as the enolase, hlyA (hemolysin), gidA (glucose-inhibited division A), vacB (virulence-associated protein B), dam (DNA adenine methyltransferase), and tagA (ToxR-regulated lipoprotein) genes, which modulated the virulence of A. hydrophila SSU (19-21, 57, 62, 64). The production of such a wide array of virulence factors by Aeromonas species is indicative of their potential to cause severe diseases in humans. These virulence factor-encoding genes might be differentially expressed in Aeromonas species depending on the environmental conditions, such as water or the human host.A cell-to-cell signaling system, known as quorum sensing (QS), might play an important role in sensing physiological conditions and helping bacteria express the virulence genes at an appropriate time under the appropriate conditions. Thus far, at least three QS circuits have been identified in Gram-negative bacteria, and they were designated LuxRI (autoinducer 1 [AI-1]), LuxS (AI-2), and AI-3 (epinephrine/norepinephrine). All of these QS systems were detected in our SSU clinical strain of A. hydrophila, and we recently demonstrated that N-acyl homoserine lactone (AHL) (AI-1) and AI-2-mediated QS controlled the virulence of A. hydrophila SSU (40, 43). Further, we observed decreased production of N-acyl homoserine lactones when we deleted two major virulence factor-encoding genes, the act gene and the gene encoding an outer membrane protein (aopB), an important component of the T3SS (65), from A. hydrophila SSU. Likewise, we observed that N-acyl homoserine lactone production was also modulated by regulatory genes, such as dam and gidA, in A. hydrophila SSU (18). Thus, differential expression of genes might also be an important factor in the pathogenesis of Aeromonas species.The presence of any virulence gene in strains of Aeromonas isolated from water should be carefully scrutinized, as such genes could be expressed better in a human host, which could lead to devastating outcomes. In addition, it is possible that in the environment certain Aeromonas clones may predominate and cause human diseases more frequently than other clones. Thus, it is important to determine the clonal variation of a range of Aeromonas species isolated from various sources and identify predominant clones by a polyphasic approach that includes biochemical phenotyping, virulence marker detection, and molecular fingerprinting techniques.In the present study, we compared 199 Aeromonas isolates, 146 of which were from water sources and 53 of which were from human patients with diarrhea in the Unites States. In addition, 28 reference and classical strains that were obtained from various culture collections and/or were isolated from specimens obtained in diverse geographical areas of the world, including water and clinical specimens, were also characterized. All isolates were biochemically identified to the phenospecies group level, examined for the presence of a set of 11 virulence factors by using DNA colony blot hybridization, and fingerprinted by using pulsed-field gel electrophoresis (PFGE). Some of the virulence factors selected, including T6SS effectors, were also examined by using functional assays. Our data provide the first suggestive evidence of water-to-human transmission, i.e., of successful colonization and infection by particular strains of certain Aeromonas species.