Use of a bacterial antimicrobial resistance gene microarray for the identification of resistant Staphylococcus aureus

As diagnostic and surveillance activities are vital to determine measures needed to control antimicrobial resistance (AMR), new and rapid laboratory methods are necessary to facilitate this important effort. DNA microarray technology allows the detection of a large number of genes in a single reaction. This technology is simple, specific and high-throughput. We have developed a bacterial antimicrobial resistance gene DNA microarray that will allow rapid antimicrobial resistance gene screening for all Gram-positive and Gram-negative bacteria. A prototype microarray was designed using a 70-mer based oligonucleotide set targeting AMR genes of Gram-negative and Gram-positive bacteria. In the present version, the microarray consists of 182 oligonucleotides corresponding to 166 different acquired AMR gene targets, covering most of the resistance genes found in both Gram-negative and -positive bacteria. A test study was performed on a collection of Staphylococcus aureus isolates from milk samples from dairy farms in Québec, Canada. The reproducibility of the hybridizations was determined, and the microarray results were compared with those obtained by phenotypic resistance tests (either MIC or Kirby-Bauer). The microarray genotyping demonstrated a correlation between penicillin, tetracycline and erythromycin resistance phenotypes with the corresponding acquired resistance genes. The hybridizations showed that the 38 antimicrobial resistant S. aureus isolates possessed at least one AMR gene.     

Virulence gene regulation in pathogenic Escherichia coli.

The ability to regulate gene expression throughout the course of an infection is important for the survival of a pathogen in the host. Thus, virulence gene expression responds to environmental signals in many complex ways. Frequently, global regulatory factors associated with specific regulators co-ordinate expression of virulence genes. In this review, we present well-described regulatory mechanisms used to co-ordinate the expression of virulence factors by pathogenic Escherichia coli with a relative emphasis on diseases caused by E. coli in animals. Many of the virulence-associated genes of pathogenic E. coli respond to environmental conditions. The involvement of global regulators, including housekeeping regulons and virulence regulons, specific regulators and then sensor regulatory systems involved in virulence, is described. Specific regulation mechanisms are illustrated using the regulation of genes encoding for fimbriae, curli, haemolysin and capsules as examples.     

Interaction with pig ileal explants of Escherichia coli O45 isolates from swine with postweaning diarrhea.

We have previously observed that Escherichia coli O45 isolates from swine postweaning diarrhea (PWD) induced attaching-effacing (A/E) lesions in experimentally inoculated gnotobiotic piglets. In the present work, ileal explant culture has been used as an in vitro model for the study of the development of A/E lesions due to these isolates. The characteristic intimate bacterial attachment and microvilli effacement with cupping and pedestal formation, identical to that observed in gnotobiotic piglets, was demonstrated in pig ileal explants inoculated with O45 E. coli isolates. The initial attachment of bacteria to the enterocytes was observed from 2 to 4 h postinoculation (PI) and full development of A/E lesions was observed within 8 h PI. In this model, we observed that 22 of 25 eaeA-positive O45 isolates induced A/E lesions. However, A/E lesions were not observed for any of 7 eaeA-negative O45 isolates. Thus, we describe a useful in vitro model for the study of A/E capacity of porcine E. coli. Use of this model has enabled us to demonstrate the relatedness of the eaeA gene to A/E capability among porcine O45 E. coli from PWD.     

Multiparametric analysis of diversity in <Emphasis Type="Italic">Botrytis cinerea</Emphasis> isolates from Israel

The necrotrophic fungus Botrytis cinerea, known as the causal agent of gray mold, is ranked second for its phytopathological global-impact. The disease is controlled by cultural means and fungicides, however, these techniques have variable effect on different fungal isolates due to the plasticity of the pathogen populations. Here we studied and characterized the genetic and virulence associated variability of B. cinerea isolates from different sources. Starting from initial survey of 31 B. cinerea isolates, collected from seven hosts in different locations of Israel, we have focused on 10 isolates that exhibited potential phytopathogenic variability. Two genetic markers (microsatellite) Bc1 and Bc7, were able to differentiate between these isolates. Our analysis demonstrates significant variability in saprophytic growth rate, necrotrophic growth rate on tomato leaves and stems, and in the incidence of infection on leaves of whole plants. Following the observation of significant correlation between saprophytic growth rate, and necrotrophic growth on leaves, we have studied normalized (by saprophytic growth) virulence. Utilization of normalized necrotrophic growth rate enabled to indicate on the presence of virulence mechanisms other than growth rate, for several isolates. Exploration of this direction illustrated variability in resistance to paraquat (associated with resistance to oxidation), which was associated with high and low superoxide dismutase (SOD) gene expression for selected isolates showing high or low paraquat resistance, respectively. Finally, we have used unsupervised learning (clustering analysis) to explore patterns in the multivariable space, which demonstrated two modes of pathogenicity in the tested B. cinerea isolates.     

Induced resistance to foliar diseases by soil solarization and Trichoderma harzianum

The effect of soil solarization and Trichoderma harzianum on induced resistance to grey mould (Botrytis cinerea) and powdery mildew (Podosphaera xanthii) was studied. Plants were grown in soils pretreated by solarization, T. harzianum T39 amendment or both, and then their leaves were inoculated with the pathogens. There was a significant reduction in grey mould in cucumber, strawberry, bean and tomato, and of powdery mildew in cucumber, with a stronger reduction when treatments were combined. Bacillus, pseudomonad and actinobacterial communities in the strawberry rhizosphere were affected by the treatments, as revealed by denaturing gradient gel electrophoresis fingerprinting. In tomato, treatments affected the expression of salicylic acid (SA)‐, ethylene (ET)‐ and jasmonic acid (JA)‐responsive genes. With both soil treatments, genes related to SA and ET – PR1a, GluB, CHI9 and Erf1 – were downregulated whereas the JA marker PI2 was upregulated. Following soil treatments and B. cinerea infection, SA‐, ET‐, and JA‐related genes were globally upregulated, except for the LOX genes which were downregulated. Upregulation of the PR genes PR1a, GluB and CHI9 in plants grown in solarized soil revealed a priming effect of this treatment on these genes' expression. The present study demonstrates the capacity of solarization and T. harzianum to systemically induce resistance to foliar diseases in various plants. This may be due to either a direct effect on the plant or an indirect one, via stimulation of beneficial microorganisms in the rhizosphere.     

Genomic relatedness among Actinobacillus pleuropneumoniae field strains of sterotypes 1 and 5 isolated from healthy and diseased pigs.

Forty-four Actinobacillus pleuropneumoniae isolates recovered from both healthy and diseased pigs were characterized by random amplified polymorphic DNA analysis (RAPD), pulsed field gel electrophoresis (PFGE) and apx toxin gene typing. Nine RAPD types and 14 PFGE patterns were identified. No common RAPD or PFGE patterns were found between strains of serotype 1 and those of serotype 5. The RAPD analysis indicated that the 15 serotype 1 strains isolated from diseased pigs were assigned to 4 RAPD types, with 66% of strains characterized by the same RAPD type. By contrast, the 5 strains of serotype 1 isolated from healthy carriers were dispersed in 4 RAPD types. These data suggest that the diversity of strains isolated from healthy pigs could be higher than that of strains recovered from diseased pigs. In addition, all serotype 5 strains exhibited a unique RAPD type. Unlike RAPD, PFGE analysis allowed discrimination among isolates of serotype 1 and among those of serotype 5. All but 3 isolates showed the same apx genotype as their respective serotype reference strain. These data indicate that RAPD analysis is a valuable rapid tool for routine subtyping of strains of serotype 1. For strains of serotype 5, a combination of several typing methods, such as PFGE and apx gene typing, is needed to provide useful information on the molecular epidemiology of swine pleuropneumonia.     

牦牛性激素结合蛋白基因克隆及组织表达研究

试验旨在阐明牦牛性激素结合蛋白（SHBG）基因CDS序列及其在母牦牛生殖轴中的表达特点,为探讨该基因在牦牛繁殖中的调控作用奠定基础。试验分别采集5头健康成年母牦牛和母黄牛的下丘脑、脑垂体前叶、输卵管、卵巢和子宫组织,根据NCBI公布的黄牛SHBG基因设计特异性扩增引物,分别采用RT-PCR技术、生物信息学方法和实时荧光定量PCR技术进行牦牛SHBG基因克隆、序列分析和组织表达检测。结果显示,牦牛SHBG核苷酸序列的编码区（CDS）全长为1 206 bp,共编码401个氨基酸,其中,亮氨酸（L）、甘氨酸（G）、脯氨酸（P）和丝氨酸（S）较多,含量分别为16.5%、9.7%、8.7%和9.0%。蛋白质分子式为C₁₉₄₄H₃₀₆₁N₅₃₅O₅₆₆S₁₀,分子质量为43.30 ku,理论等电点5.63,与黄牛核苷酸和氨基酸同源性分别为99.0%和97.5%。SHBG蛋白为亲水不稳定蛋白,存在跨膜结构和信号肽;二级结构中主要包含无规则卷曲和延伸链,与三级结构分析一致。系统进化树表明牦牛与黄牛亲缘关系最近。SHBG基因在牦牛输卵管的表达量显著高于其他组织（P<0.05）,其他组织之间差异不显著（P>0.05）。SHBG基因在黄牛输卵管表达量极显著高于牦牛（P<0.01）,在黄牛卵巢表达量显著高于牦牛（P<0.05）,两物种的其他组织中表达量差异不显著（P>0.05）。本研究探讨了牦牛SHBG基因序列及其在生殖轴的表达特性,为进一步研究SHBG基因对牦牛繁殖调控作用提供了一定理论依据。     

Regenerating fish optic nerves and a regeneration-like response in injured optic nerves of adult rabbits

Regeneration of fish optic nerve (representing regenerative central nervous system) was accompanied by increased activity of regeneration-triggering factors produced by nonneuronal cells. A graft of regenerating fish optic nerve, or a "wrap-around" implant containing medium conditioned by it, induced a response associated with regeneration in injured optic nerves of adult rabbits (representing a nonregenerative central nervous system). This response was manifested by an increase of general protein synthesis and of selective polypeptides in the retinas and by the ability of the retina to sprout in culture.     

The inherent variability of water stress indicators in apple, nectarine and pear orchards, and the validity of a leaf-selection procedure for water potential measurements

The following two topics were examined: (1) The variability in the measurement of leaf water potential (LWP), stem water potential (SWP), maximum daily trunk shrinkage (MDS), and soil water tension (SWT) in apple, nectarine and pear orchards; and (2) The validity of a leaf-selection procedure for SWP measurements in commercial apple orchards. 27 trees were selected in an apple orchard, 27 in a nectarine orchard, and 30 in a pear orchard. The trees were close to each other. The measurements comprised of: midday SWP in apple, nectarine and pear; midday LWP in apple; MDS in apple and nectarine; and SWT in pear. The mean and standard errors (SEs) of each water status indicator in each species were calculated for an increasing sample size. The sample sizes required for stable averages were: SWP – 4, 5, and 8 trees for apple, nectarine and pear, respectively; MDS – 17 and 16 trees for apple and nectarine, respectively; SWT – 21 for pear trees. The relative SEs (i.e. percent of population mean) were 2.4, 6.1 and 10.1% in SWP/LWP, MDS and SWT, respectively. Possible explanations for the differing variability of the various water status indicators are discussed. The results show that smaller samples were sufficient to represent SWP and LWP properly than what was required for MDS and SWT. 9 commercial apple plots were selected and about 25 randomly selected leaves were used for midday SWP measurements in each plot (i.e. experimental sets). About 5 leaves on closely adjacent “representative” trees were selected in each of the commercial plots (i.e. commercial sets) and midday SWP was measured. The average difference in SWP between the experimental and the commercial sets was –0.127 MPa. The choice of closely adjacent trees increased the deviation from the experimental sets. The use of a reasonable sample size (n=7) may enable midday SWP to be measured within ±0.15 MPa in most commercial orchards.     

The effects of pre-harvest dehydration on the composition of once-over harvested sweet paprika

When paprika fruits (Capsicum annuum L.) were allowed to dry in the sun, on the plant itself, before harvest, there was a considerable increase in the dry matter content of the subsequently harvested fruit, along with a decrease in the total fresh yield. The dry matter yield did not vary during the drying period, but the colour intensity of the fruit increased continuously. Early cessation of irrigation accelerated the drying rate and intensified fruit colour, but lowered the total dry matter yield.Removal of seeds, placenta parts and stalk after harvest increased fruit colour intensity but lowered yields.Leaving fruit to dry in the sun before harvest resulted in a considerable saving in transport and storage volume, as well in the amount of energy required otherwise to dry the fruit in the dehydration plant. It also opens up possibilities of once-over mechanized harvesting of high quality fruit.