Metabolism of acrylonitrile by Klebsiella pneumoniae

A gram-negative rod-shaped bacterium capable of utilizing acrylonitrile as the sole source of nitrogen was isolated from industrial sewage and identified as Klebsiella pneumoniae. The isolate was capable of utilizing aliphatic nitriles containing 1 to 5 carbon atoms or benzonitrile as the sole source of nitrogen and either acetamide or propionamide as the sole source of both carbon and nitrogen. Gas chromatographic and mass spectral analyses of culture filtrates indicated that K. pneumoniae was capable of hydrolyzing 6.15 mmol of acrylonitrile to 5.15 mmol of acrylamide within 24 h. The acrylamide was hydrolyzed to 1.0 mmol of acrylic acid within 72 h. Another metabolite of acrylonitrile metabolism was ammonia, which reached a maximum concentration of 3.69 mM within 48 h. Nitrile hydratase and amidase, the two hydrolytic enzymes responsible for the sequential metabolism of nitrile compounds, were induced by acrylonitrile. The optimum temperature for nitrile hydratase activity was 55°C and that for amidase was 40°C; both enzymes had pH optima of 8.0.Abbreviations PBM phosphate buffered medium - GC gas chromatography - GC/MS gas chromatography/mass spectrometry     

Influence of Cellular Factors on Immune Unresponsiveness Induced by Klebsiella pneumoniae Capsular Antigen

The mechanisms by which immune unresponsiveness (immune paralysis) develops are still uncertain. The present work was based on the assumption that this condition may be due to failure of certain activities of the macrophages. Data from passive transfer of such cells are interpreted as supporting this hypothesis. Purified capsular polysaccharide from type 2 Klebsiella pneumoniae induced immunity when given to Swiss albino mice in 5-mug amounts and immune paralysis when given in 1,000-mug amounts. The unresponsive state lapsed to that of acquired immunity 3 months after induction. Passive transfer of cells from groups of K. pneumoniae immune, "paralyzed," and control mice showed that peritoneal cells from "paralyzed" donors induced significant protection against challenge in both control and "paralyzed" recipients. In contrast, spleen cells from control, "paralyzed," and immune animals failed to effect such transfer. The finding that unresponsiveness could be terminated by peritoneal cells and not spleen cells indicates that macrophages played a primary role in immune paralysis, possibly owing to their loss of capacity to transfer factors or information for induction of antibody synthesis.     

Reduction of fensulfothion to fensulfothion sulfide by Klebsiella pneumoniae.

A cell suspension of Klebsiella pneumoniae converted the organophosphorus pesticide fensulfothion to a product that was shown by chemical oxidation, gas-liquid chromatography, infrared spectrophotometry, and mass spectrometry to be fensulfothion sulfide. Further alteration of this metabolite was not noted.     

Reduction of fensulfothion to fensulfothion sulfide by Klebsiella pneumoniae.

A cell suspension of Klebsiella pneumoniae converted the organophosphorus pesticide fensulfothion to a product that was shown by chemical oxidation, gas-liquid chromatography, infrared spectrophotometry, and mass spectrometry to be fensulfothion sulfide. Further alteration of this metabolite was not noted.     

Hemolytic activity of Klebsiella pneumoniae and Klebsiella oxytoca

We demonstrated that Klebsiella pneumoniae and Klebsiella oxytoca possess a selective haemolytic activity on rabbit erythrocytes. Thirty one Klebsiella strains (18 strains of K. pneumoniae and 13 strains of K. oxytoca) were isolated from hospitalized patients. The liquid (Trypcase-soy broth--TSB) and solid (Trypcase-soy agar--TSA) medium, containing the red cells were used for the tests. All the screened strains showed a haemolytic effect on rabbit erythrocytes, provided that the supernatants of the cultures were preincubated with beta-mercaptoethanol or calcium chloride. There was no human and sheep erythrocyte lysis.     

Extracellular polysaccharide production by Klebsiella pneumoniae and its relationship to virulence

Klebsiella pneumoniae serotype 1 and serotype 2 and their capsular variants were examined for production of cell-associated capsular polysaccharides and extracellular capsular polysaccharides. The virulence of these organisms in experimental animals was examined via intraperitoneal injection in mice and transtracheal inoculation into the lungs of rats. It was found that the production of either polysaccharide component correlated with the observed virulence. The extracellular polysaccharides were purified by ethanol precipitation, electrodialysis, extraction with quaternary ammonium salts, and gel filtration. These purification steps allowed for the separation and purification of both the extracellular lipopolysaccharide and the extracellular capsular polysaccharide. Purified extracellular capsular polysaccharide and extracellular lipopolysaccharide were co-injected with K. pneumoniae intraperitoneally into mice to determine if either of these substances would produce an effect on the natural course of infection in these animals. These studies showed that only purified extracellular lipopolysaccharide enhanced the virulence of K. pneumoniae when co-injected into mice, and this virulence enhancement correlated with the content of extracellular lipopolysaccharide, but not extracellular capsular polysaccharide in mixtures of these polysaccharides. Saponification of K. pneumoniae serotype 1 extracellular polysaccharides significantly decreased their virulence-enhancing capabilities in mice, further suggesting that extracellular lipopolysaccharide may play a role in these infections.     

Klebsiella pneumoniae survives within macrophages by avoiding delivery to lysosomes

Klebsiella pneumoniae is an important cause of community‐acquired and nosocomial pneumonia. Evidence indicates that Klebsiella might be able to persist intracellularly within a vacuolar compartment. This study was designed to investigate the interaction between Klebsiella and macrophages. Engulfment of K. pneumoniae was dependent on host cytoskeleton, cell plasma membrane lipid rafts and the activation of phosphoinositide 3‐kinase (PI3K). Microscopy studies revealed that K. pneumoniae resides within a vacuolar compartment, the Klebsiella‐containing vacuole (KCV), which traffics within vacuoles associated with the endocytic pathway. In contrast to UV‐killed bacteria, the majority of live bacteria did not co‐localize with markers of the lysosomal compartment. Our data suggest that K. pneumoniae triggers a programmed cell death in macrophages displaying features of apoptosis. Our efforts to identify the mechanism(s) whereby K. pneumoniae prevents the fusion of the lysosomes to the KCV uncovered the central role of the PI3K–Akt–Rab14 axis to control the phagosome maturation. Our data revealed that the capsule is dispensable for Klebsiella intracellular survival if bacteria were not opsonized. Furthermore, the environment found by Klebsiella within the KCV triggered the down‐regulation of the expression of cps. Altogether, this study proves evidence that K. pneumoniae survives killing by macrophages by manipulating phagosome maturation that may contribute to Klebsiella pathogenesis.     

Fermentation of glycerol to 1,3-propanediol and 2,3-butanediol by Klebsiella pneumoniae

Klebsiella pneumoniae was shown to convert glycerol to 1,3-propanediol, 2,3-butanediol and ethanol under conditions of uncontrolled pH. Formation of 2,3-butanediol starts with some hours' delay and is accompanied by a reuse of the acetate that was formed in the first period. The fermentation was demonstrated in the type strain of K. pneumoniae, but growth was better with the more acid-tolerant strain GT1, which was isolated from nature. In continuous cultures in which the pH was lowered stepwise from 7.3 to 5.4, 2,3-butanediol formation started at pH 6.6 and reached a maximum yield at pH 5.5, whereas formation of acetate and ethanol declined in this pH range. 2,3-Butanediol and acetoin were also found among the products in chemostat cultures grown at pH 7 under conditions of glycerol excess but only with low yields. At any of the pH values tested, excess glycerol in the culture enhanced the butanediol yield. Both effects are seen as a consequence of product inhibition, the undissociated acid being a stronger trigger than the less toxic diols and acid anions. The possibilities for using the fermentation type described to produce 1,3-propanediol and 2,3-butanediol almost without by-products are discussed. Received: 4 February 1998 / Received revision: 30 March 1998 / Accepted: 13 April 1998     

Metabolism of benzonitrile and butyronitrile by Klebsiella pneumoniae.

A strain of Klebsiella pneumoniae that used aliphatic nitriles as the sole source of nitrogen was adapted to benzonitrile as the sole source of carbon and nitrogen. Gas chromatographic and mass spectral analyses of culture filtrates indicated that K. pneumoniae metabolized 8.4 mM benzonitrile to 4.0 mM benzoic acid and 2.7 mM ammonia. In addition, butyronitrile was metabolized to butyramide and ammonia. The isolate also degraded mixtures of benzonitrile and aliphatic nitriles. Cell extracts contained nitrile hydratase and amidase activities. The enzyme activities were higher with butyronitrile and butyramide than with benzonitrile and benzamide, and amidase activities were twofold higher than nitrile hydratase activities. K. pneumoniae appears promising for the bioremediation of sites contaminated with aliphatic and aromatic nitriles.     

Metabolism of benzonitrile and butyronitrile by Klebsiella pneumoniae.

A strain of Klebsiella pneumoniae that used aliphatic nitriles as the sole source of nitrogen was adapted to benzonitrile as the sole source of carbon and nitrogen. Gas chromatographic and mass spectral analyses of culture filtrates indicated that K. pneumoniae metabolized 8.4 mM benzonitrile to 4.0 mM benzoic acid and 2.7 mM ammonia. In addition, butyronitrile was metabolized to butyramide and ammonia. The isolate also degraded mixtures of benzonitrile and aliphatic nitriles. Cell extracts contained nitrile hydratase and amidase activities. The enzyme activities were higher with butyronitrile and butyramide than with benzonitrile and benzamide, and amidase activities were twofold higher than nitrile hydratase activities. K. pneumoniae appears promising for the bioremediation of sites contaminated with aliphatic and aromatic nitriles.     

MALDI-TOF MS用于肺炎克雷伯菌同源性分析的初步研究

目的评价基质辅助激光解吸电离飞行时间质谱(matrix-assisted laser desorption/ionization-time of flight mass spectrometry,MALDI-TOF MS)对肺炎克雷伯菌(Klebsiella pneumoniae,KPN)同源性分析的能力。方法对2013年2-4月青岛大学附属医院重症监护病房(ICU)分离的7株KPN和其他科室分离的13株KPN进行溯源性回顾分析,采用方法为脉冲场凝胶电泳(pulsed-field gel electrophoresis,PFGE)、多位点序列分型(multilocus sequence typing,MLST)和MALDI-TOF MS。结果 PFGE和MLST结果一致,MALDI-TOF MS同源性分析中将20株菌株分为2类,Ⅰ类为来自ICU的7株菌,Ⅱ类为其他科室的13株菌,与前2种方法得到的结果基本一致。结论 MALDI-TOF MS技术能够准确鉴定肺炎克雷伯菌且可对其进行同源性分析,较其他同源性分析方法快捷、方便,可满足临床对院感工作的需求。     

Killing of Klebsiella pneumoniae by human alveolar macrophages

We investigated putative mechanisms by which human surfactant protein A (SP-A) effects killing of Klebsiella pneumoniae by human alveolar macrophages (AMs) isolated from bronchoalveolar lavagates of patients with transplanted lungs. Coincubation of AMs with human SP-A (25 microg/ml) and Klebsiella resulted in a 68% decrease in total colony forming units by 120 min compared with AMs infected with Klebsiella in the absence of SP-A, and this SP-A-mediated effect was abolished by preincubation with N(G)-monomethyl-L-arginine. Incubation of transplant AMs with SP-A increased intracellular Ca(2+) concentration ([Ca(2+)](i)) by 70% and nitrite and nitrate (NO(x)) production by 45% (from 0.24 +/- 0.02 to 1.3 +/- 0.21 nmol small middle dot 10(6) AMs(-1).h(-1)). Preincubation with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester inhibited the increase in [Ca(2+)](i) and abrogated the SP-A-mediated Klebsiella phagocytosis and killing. In contrast, incubation of AMs from normal volunteers with SP-A decreased both [Ca(2+)](i) and NO(x) production and did not result in killing of Klebsiella. Significant killing of Klebsiella was also seen in a cell-free system by sustained production of peroxynitrite (>1 microM/min) at pH 5 but not at pH 7.4. These findings indicate that SP-A mediates pathogen killing by AMs from transplant lungs by stimulating phagocytosis and production of reactive oxygen-nitrogen intermediates.     

Hydrophobic and hemagglutinating properties of Klebsiella pneumoniae and Klebsiella oxytoca

The aim of this study was to estimated hydrophobic and hemnagglutinating properties of Klebsiella pneumoniae and Klebsiella oxytoca rods. The hydrophobcity was evaluated according to the method of Rosenberg et al. The hemagglutinating properties were estimated by method of Blanco et al. Forty seven hydrophobic Klebsiella strains (30 K. pneumoniae strains and 17 K. oxytoca strains) were detected. Hemagglutinating properties were observed in 65 Klebsiella strains (45 K. pneumoniae strains and 20 K. oxytoca strains). Hemagglutination of sheep tannined erythrocytes the most frequently was observed. Inhibition of hemagglutination of erythrocytes by K. pneumoniae strains was most frequently observed in presence of D-glucose and D-mannose and by K. oxytoca strains in presence of D-glucose.