Providencia alcalifaciens causes barrier dysfunction and apoptosis in tissue cell culture: potent role of lipopolysaccharides on diarrheagenicity

Providencia alcalifaciens is a member of the Enterobacteriaceae family that occasionally causes diarrheagenic illness in humans via the intake of contaminated foods. Despite the epidemiological importance of P. alcalifaciens, little is known about its pathobiology. Here we report that P. alcalifaciens causes barrier dysfunction in Caco-2 cell monolayers and induces apoptosis in calf pulmonary artery endothelial cells. P. alcalifaciens infection caused a 30% reduction in transepithelial resistance in Caco-2 cell monolayers, which was greater than that for cells infected with Shigella flexneri or non-pathogenic Escherichia coli. As with viable bacteria, bacterial lysates treated with heat, benzonase or proteinase, but not with polymixin B, were also involved in the cellular response. TLR4 antibody neutralisation significantly restored the P. alcalifaciens-induced transepithelial resistance reduction in Caco-2 cells, suggesting that lipopolysaccharides (LPSs) might play a central role in this cellular response. Western blotting further indicated that P. alcalifaciens LPSs reduced occludin levels, whereas LPSs from Shigella or E. coli did not. Although the viability of Caco-2 cells was not altered significantly, the calf pulmonary artery endothelial cell line was highly sensitive to P. alcalifaciens infection. This sensitivity was indeed dependent on LPS, which induced rapid apoptosis. Together, these data show that P. alcalifaciens LPSs participate in epithelial barrier dysfunction and endothelial apoptosis. The findings give insight into the LPS-dependent cell signal events affecting diarrheagenicity during infection with P. alcalifaciens.     

Pseudopterosin Content Variability of the Purple Sea Whip Pseudopterogorgia elisabethae at the Islands of San Andres and Providencia (Sw Caribbean)

To determine pseudopterosin composition and concentration in colonies of Pseudopterogorgia elisabethae from the islands of San Andres and Providencia, we collected fragments of individual colonies at various sites and depth ranges around the islands. Chromatographic profiles of the polar fraction, particularly those obtained by HPLC-MS analyses, allowed us to recognize two different chemotypes. Chemotype 1 characterized samples from Providencia whereas chemotype 2 characterized samples from San Andres. A complex pseudopterosin mixture (compounds 1-13) characterized chemotype 1. These compounds were isolated by a combination of chromatographic methods and identified by spectroscopic methods (MS, UV, 1H, and 13C NMR). We identified the known pseudopterosins G and K and seco-pseudopterosin A. We also isolated and identified seven new compounds, pseudopterosins P-V, isomers of known pseudopterosins. Pseudopterosins G and K were found at concentrations ranging between 1 and 3% of the animal dry mass. Pseudopterosins Q and U were the major compounds reaching up to 6% of the animal dry mass at some locations. Major metabolites in chemotype 2 had a molecular weight and fragmentation pattern different from that observed in the pseudopterosins, as determined by HPLC-MS. Total pseudopterosin concentration in this chemotype was below 3% dry mass at all sites. Total pseudopterosin concentration was significantly higher in chemotype 1, with concentrations ranging between 4 and 20% dry mass. At most locations on Providencia, however, total pseudopterosin concentration ranged between 11 and 15% dry mass. Concentrations exceed reports from other locations in the Caribbean. Furthermore, pseudopterosin composition in our samples is quite different from those in specimens of P. elisabethae from the Bahamas and Bermuda. Pseudopterosins G, K, and P-V are characteristic of P. elisabethae colonies from the island of Providencia, while pseudopterosins A-D are characteristic of colonies of P. elisabethae from the Bahamas islands, and pseudopterosins E-L have been isolated from P. elisabethae from the Bahamas and Bermuda. The overall morphology of P. elisabethae can be variable, and chemical differences are not correlated to specific morphs. We confirmed the species identity of each colony by morphological and sclerite analysis and found no significant differences in sclerite dimensions among different colonies and chemotypes.     

食源性普罗威登斯菌的分离鉴定和耐药性研究

本文研究了食源性普罗威登斯菌在肉类食品中的的分布,以及其耐药表型与I型整合子的携带状况。本文采集了市售猪肉、鸡肉和牛肉等肉类食品,对普罗威登斯菌进行分离与鉴定;采用纸片扩散法对已分离鉴定的普罗威登斯菌进行药敏实验;利用聚合酶链式反应技术筛选携带I型整合子的菌株。结果表明,85份样品中,有38份样品检出普罗威登斯菌,检出率达44.70%。38株普罗威登斯菌中,有44.74%的分离株是多重耐药菌株,最多耐6种抗生素。有两株普罗威登斯菌携带I型整合酶,一株拉氏普罗威登斯菌携带耐药基因盒。这是在国内肉类食品分离的普罗威登斯菌中首次发现I型整合子阳性菌株,表明食品中这种携带有多重耐药的菌株有可能通过食物链向人类传播,是对人类健康造成威胁的潜在危险因素。     

铜抗性菌株的筛选及其对Cu2+的吸附性能

利用选择性培养基分离出铜抗性细菌BX,经鉴定该菌为产碱普罗威登斯菌(Providencia alcalifaciens)。考察了细菌BX对环境的适应性,讨论了pH、初始Cu²⁺浓度等对其吸附铜离子的影响,分析了吸附过程的动力学及等温吸附特性,并以多孔陶瓷为载体对其进行固定化。结果表明,该菌对Cu²⁺和NaCl的抗性浓度分别为7 mmol·L^-1和7.5%,可生长于pH4.0～11.0、15～50℃的环境中;其最佳吸附条件为pH5.5、温度30℃、起始Cu²⁺浓度100 mg·L^-1,在该条件下,Cu²⁺吸附率达85.84%,吸附量为128.74 mg·g^-1;其对Cu²⁺的吸附符合准二级动力学方程和Freundlich等温吸附模型;采用曝气挂膜法将细菌固定于多孔陶瓷上,形成的菌膜对50 mg·L^-1铜离子的吸附率达92.53%。表明细菌BX对Cu²⁺有较强的吸附能力,对含Cu²⁺废水的处理具有良好的应用前景。     

普罗威斯登菌对钯(Ⅱ)的吸附特性研究

将普罗威斯登菌作为吸附剂来研究其对钯(Ⅱ)的吸附特性,探讨了pH、离子强度对吸附效果的影响以及吸附过程中的菌体活性和离子交换机制。结果发现,pH和离子强度对生物吸附的影响很大,菌体表面的钙、镁和钾等阳离子和钯(Ⅱ)离子发生了非化学计量的离子交换。另外,生物吸附与菌体活性无关,吸附过程中绝大部分菌体死亡。     

Substrate specificity of a recombinant d-lyxose isomerase from Providencia stuartii for monosaccharides

The specific activity and catalytic efficiency (k_cat/K_m) of the recombinant putative protein from Providencia stuartii was the highest for d-lyxose among the aldose substrates, indicating that it is a d-lyxose isomerase. Gel filtration analysis suggested that the native enzyme is a dimer with a molecular mass of 44 kDa. The maximal activity for d-lyxose isomerization was observed at pH 7.5 and 45 °C in the presence of 1 mM Mn²⁺. The enzyme exhibited high isomerization activity for aldose substrates with the C2 and C3 hydroxyl groups in the left-hand configuration, such as d-lyxose, d-mannose, l-ribose, d-talose, and l-allose (listed in decreasing order of activity). The enzyme exhibited the highest activity for d-xylulose among all pentoses and hexoses. Thus, d-lyxose was produced at 288 g/l from 500 g/l d-xylulose by d-lyxose isomerase at pH 7.5 and 45 °C for 2 h, with a conversion yield of 58 % and a volumetric productivity of 144 g l^− 1 h^− 1. The observed k_cat/K_m (920 mM^− 1 s^− 1) of P. stuartiid-lyxose isomerase for d-xylulose is higher than any of the k_cat/K_m values previously reported for sugar and sugar phosphate isomerases with monosaccharide substrates. These results suggest that the enzyme will be useful as an industrial producer of d-lyxose.     

普罗维登斯菌和希瓦氏菌对Pt(IV)的吸附特性

研究了普罗维登斯菌和希瓦氏菌两种微生物对Pt(IV)的吸附特性。pH和离子强度条件优化实验结果表明,pH=2.0时吸附效果较好,吸附量分别为58.62和72.20 mg/g;随着离子强度的增加,普罗维登斯菌对Pt(IV)的吸附量增加而希瓦氏菌却降低;Pt(IV)和Pd(II)共存时,两种微生物吸附剂均优先吸附Pd(II)。动力学和等温吸附实验结果表明,普罗维登斯菌吸附Pt(IV)的过程更符合拟二级动力学模型和Langmuir等温模型,说明化学吸附是该过程的限速步骤,且为单分子层吸附,其理论最大吸附量为136.10 mg/g。因此,以上研究结果表明,普罗维登斯菌和希瓦氏菌可以吸附回收溶液中的Pt(IV)离子。