Klebsiella pneumoniae haemolysin adsorption to red blood cells

It was necessary to incubate the Klebsiella pneumoniae haemolysin with erythrocytes at 37 degrees C to produce the whole lytic action. The amount of attached klebolysin at 4 degrees C increased as its concentration in the medium increased, until the erythrocyte surface was saturated. Treatment with 2-mercaptoethanol was necessary to permit the adsorption; it was inhibited by low concentrations of cholesterol. Klebsolysin was immunogenic and its antiserum neutralized its own haemolytic effect and streptolysin O. Anti-streptolysin serum also neutralized klebsolysin. Streptolysin O attachment to erythrocytes impeded the posterior klebolysin adsorption in the same way that klebsolysin adsorption interfered with streptolysin O attachment.     

Klebsiella pneumoniae haemolysin adsorption to red blood cells

It was necessary to incubate the Klebsiella pneumoniae haemolysin with erythrocytes at 37°C to produce the whole lytic action. The amount of attached klebolysin at 4°C increased as its concentration in the medium increased, until the erythrocyte surface was saturated. Treatment with 2-mercaptoethanol was necessary to permit the adsorption; it was inhibited by low concentrations of cholesterol. Klebsolysin was immunogenic and its antiserum neutralized its own haemolytic effect and streptolysin O. Anti-streptolysin serum also neutralized klebsolysin. Streptolysin O attachment to erythrocytes impeded the posterior klebolysin adsorption in the same way that klebsolysin adsorption interfered with streptolysin O attachment.     

Klebsiella pneumoniae disassembles host microtubules in lung epithelial cells

Klebsiella pneumoniae raises significant concerns to the health care industry as these microbes are the source of widespread contamination of medical equipment, cause pneumonia as well as other multiorgan metastatic infections and have gained multidrug resistance. Despite soaring mortality rates, the host cell alterations occurring during these infections remain poorly understood. Here, we show that during in vitro and in vivo K. pneumoniae infections of lung epithelia, microtubules are severed and then eliminated. This destruction does not require direct association of K. pneumoniae with the host cells, as microtubules are disassembled in cells that are distant from the infecting bacteria. This microtubule dismantling is dependent on the K. pneumoniae (Kp) gene ytfL as non‐pathogenic Escherichia coli expressing Kp ytfL disassemble microtubules in the absence of K. pneumoniae itself. Our data points to the host katanin catalytic subunit A like 1 protein (KATNAL1) and the katanin regulatory subunit B1 protein (KATNB1) as the gatekeepers to the microtubule severing event as both proteins localise specifically to microtubule cut sites. Infected cells that had either of these proteins knocked out maintained intact microtubules. Taken together, we have identified a novel mechanism that a bacterial pathogen has exploited to cause microtubule destruction within the host epithelia.     

肺炎克雷伯菌研究进展

近年来由于各种抗菌药物的广泛使用,导致肺炎克雷伯菌多重耐药现象普遍存在,给临床治疗带来极大的困扰,造成医院获得性肺炎感染严重的现状。着重论述了肺炎克雷伯菌流行现状、耐药机制、致病因子及防治措施。     

肺炎克雷伯菌生物被膜的体外模型建立

目的研究临床分离的肺炎克雷伯菌在体外形成生物被膜的情况,为进一步研究生物被膜肺炎克雷伯菌的耐药机制奠定基础。方法采用改良平板法建立肺炎克雷伯菌生物被膜模型,用喷金法和扫描电镜观察鉴定,并对生物被膜的形成进行定量分析。结果23株临床分离的肺炎克雷伯菌菌株生物被膜形成能力不同,以强阳性生物被膜形成能力者为最多数。结论绝大多数临床分离的肺炎克雷伯菌菌株具有较强的生物被膜形成能力。应用改良平板法能够较好的在体外建立其生物被膜模型。     

Genotoxic Klebsiella pneumoniae in Taiwan

Background

Colibactin is a nonribosomal peptide-polyketide synthesized by multi-enzyme complexes encoded by the pks gene cluster. Colibactin-producing Escherichia coli have been demonstrated to induce host DNA damage and promote colorectal cancer (CRC) development. In Taiwan, the occurrence of pyogenic liver abscess (PLA) has been suggested to correlate with an increasing risk of CRC, and Klebsiella pneumoniae is the predominant PLA pathogen in Taiwan

Methodology/Principal Findings

At the asn tRNA loci of the newly sequenced K. pneumoniae 1084 genome, we identified a 208-kb genomic island, KPHPI208, of which a module identical to the E. coli pks colibactin gene cluster was recognized. KPHPI208 consists of eight modules, including the colibactin module and the modules predicted to be involved in integration, conjugation, yersiniabactin production, microcin production, and unknown functions. Transient infection of BALB/c normal liver cells with K. pneumoniae 1084 increased the phosphorylation of histone H2AX, indicating the induction of host DNA damage. Colibactin was required for the genotoxicity of K. pneumoniae 1084, as it was diminished by deletion of clbA gene and restored to the wild type level by trans-complementation with a clbA coding plasmid. Besides, BALB/c mice infected with K. pneumoniae 1084 exhibited enhanced DNA damage in the liver parenchymal cells when compared to the isogenic clbA deletion mutant. By PCR detection, the prevalence of pks-positive K. pneumoniae in Taiwan is 25.6%, which is higher than that reported in Europe (3.5%), and is significantly correlated with K1 type, which predominantly accounted for PLA in Taiwan.

Conclusions

Our knowledge regarding how bacteria contribute to carcinogenesis has just begun. The identification of genotoxic K. pneumoniae and its genetic components will facilitate future studies to elucidate the molecular basis underlying the link between K. pneumoniae, PLA, and CRC.     

Fimbrial hemagglutinins in Klebsiella pneumoniae

Eleven Klebsiella pneumoniae strains were isolated from urine specimens which were examined for their ability to produce hemagglutinins (HAs). Bacteria were grown under various culture conditions. Suspension of bacteria grown in broth or on Phosphate-buffered nutrient agar (PBA) were tested for agglutination in the presence and absence of 2% (w/v) D-mannose, on rockedtiles at 4 degrees C and ambient temperature with mangroup-O(M), fowl(F), ox(O), guinea-pig(G), horse (H), rabbit (R) and sheep (S) erythrocytes and tannic acid treated, but not fresh oxen erythrocytes. Each of the 11 strains was hemagglutinating. Ten strains (99%) producing two or three hemagglutinins (HAs), were multiple hemagglutinating. One strain produced only mannose-resistant, Klebsiella, the "Tanned ox hemagglutinin" (MR/K-HA). Solely mannose-sensitive hemagglutinin (MS/HA) was not produced by any of the strains. No mannose-sensitive hemagglutinating strains acted on sheep erythrocytes. Three main kinds of hemagglutinin (HA) were detected. These were: (a) a mannose-sensitive hemagglutinin (MS-HA); (b) a mannose resistant, Klebsiella, the "tanned ox hemagglutinin (MR/K-HA); (c) mannose-resistant, Proteus hemagglutinin (MR/P-HA). All strains (100%) produced MR/K-HA, 45% of MR/K-HA+ strains produced MR/P-HA at 37 degrees C and 99% of all strains produced MS/HA, MR/P-HA activity was never dependent on MR/K-HA: Electronmicroscopic examination of bacteria showed that all strains were fimbriate.     

Selective adhesion of mast cells to tracheal epithelial cells in vitro

In allergic and nonallergic lung diseases, if intraluminal mast cells adhere to airway epithelium, inflammatory mediators released from activated mast cells may reach high local concentrations and thus greatly affect airway function. To determine whether mast cells adhere to airway epithelial cells, radiolabeled or unlabeled dog mastocytoma cells were incubated with cultured dog tracheal epithelial cells, with extracellular matrix substrates, and with cryostat-cut sections of dog trachea. Mast cells adhered well to cultured epithelial cells (35 +/- 13% adhesion, mean +/- 1 SD, n = 23) but adhered poorly to types I and IV collagen or to fibronectin (less than 7.5% mean adhesion in all cases). Similarly, in tracheal tissue sections, mast cells adhered preferentially to epithelial cells in surface epithelium or in submucosal glands but not to basal membrane or connective tissue. Adhesion to cultured epithelial cells was a characteristics of a subpopulation of mast cells, could persist for more than 48 h, did not require energy or the presence of divalent cations, and was not mediated by a known family of leukocyte-associated adhesion glycoproteins. Adhesion was completely abolished by pretreatment of mast cells with pronase E or proteinase K but not with trypsin (up to 10 micrograms/ml at 37 degrees C for 20 min each). In contrast, pretreatment of cultured epithelial cells with any of these proteinases had no effect on adhesion. It is concluded that dog mastocytoma mast cells adhere to dog tracheal epithelial cells and do so selectively. It is suggested that mast cell adhesion to airway epithelium may play a role in the effectiveness of mast cell-epithelial cell interactions, and thus, in certain lung diseases, airway function may be affected by intraluminal mast cells more than is currently appreciated.     

Cryptic growth in Klebsiella pneumoniae

Summary The ability of Klebsiella pneumoniae to grow on its own soluble lysis products is shown in a series of batch growth experiments. Maximum specific growth rate coefficients ranging from 0.69 to 1.46 h^-1 were obtained with experimental cryptic yield coefficients ranging between 0.42 to 0.52 (mg-cell-C/mg-substrate-C). These kinetic data are used to calibrate a model which demonstrates that depression of theoretical maximum yield coefficients relative to experimentally obtained values can be explained by cryptic growth phenomena without the need to resort to the use of physiologically undefined, mathematical constants. Growth of K. pneumoniae on sonicated cells derived from steady-state chemostat cultures was followed in batch culture and observed to occur with no lag phase. Batch growth curves did not indicate either diauxic or polyauxic growth, suggesting simultaneous utilization of the complex organic substrate mixture. These data suggest that cryptic growth is probably a real event occurring in growing chemostat cultures under ideal growth conditions and most probably also under starvation conditions.     

Citrate transport in Klebsiella pneumoniae

Sodium ions were specifically required for citrate degradation by suspensions of K. pneumoniae cells which had been grown anaerobically on citrate. The rate of citrate degradation was considerably lower than the activities of the citrate fermentation enzymes citrate lyase and oxaloacetate decarboxylase, indicating that citrate transport is rate limiting. Uptake of citrate into cells was also Na+ -dependent and was accompanied by its rapid metabolism so that the tricarboxylic acid was not accumulated in the cells to significant levels. The transport could be stimulated less efficiently by LiCl. Li+ ions were cotransported with citrate into the cells. Transport and degradation of citrate were abolished with the uncoupler [4-(trifluoromethoxy)phenylhydrazono]propanedinitrile (CCFP). After releasing outer membrane components and periplasmic binding proteins by cold osmotic shock treatment, citrate degradation became also sensitive towards monensin and valinomycin. The shock procedure had no effect on the rate of citrate degradation indicating that the transport is not dependent on a binding protein. Citrate degradation and transport were independent of Na+ ions in K. pneumoniae grown aerobically on citrate and in E. coli grown anaerobically on citrate plus glucose. An E. coli cit+ clone obtained by transformation of K. pneumoniae genes coding for citrate transport required Na specifically for aerobic growth on citrate indicating that the Na-dependent citrate transport system is operating. Na+ and Li+ were equally effective in stimulating citrate degradation by cell suspensions of E. coli cit+. Citrate transport in membrane vesicles of E. coli cit+ was also Na+ dependent and was energized by the proton motive force (delta micro H+). Dissipation of delta micro H+ or its components delta pH or delta psi by ionophores either totally abolished or greatly inhibited citrate uptake. It is suggested that the systems energizing citrate transport under anaerobic conditions are provided by the outwardly directed cotransport of metabolic endproducts with protons yielding delta pH and by the decarboxylation of oxaloacetate yielding delta pNa+ and delta psi. In citrate-fermenting K. pneumoniae an ATPase which is activated by Na+ was not found. The cells contain however a proton translocating ATPase and a Na+/H+ antiporter in their membrane.     

Cyclodextrin-Glucanotransferase von Klebsiella pneumoniae

1. When growing with cyclodextrins, Klebsiella pneumoniae M 5 al produces extracellular cyclodextrin glucanotransferase in amounts comparable to those obtained during the growth with potato starch.
2. Intracellular cyclodextrin glucanotransferase-activity was demonstrated to be present in the homogenates of cells grown with cyclodextrins. In addition, an amylomaltase-like enzyme and the maltodextrin phosphorylase could be pointed out. The cyclodextrins are metabolized to glucose-1-phosphate and glucose by the concerted actions of these three enzymes. paraGlucose-1-phosphate is liberated from cyclohexaamylose by the actions of purified cyclodextrin glucanotransferase and purified maltodextrin phosphorylase. The liberation of the sugar phosphate is increased fivefold by addition of glucose as an acceptor. This sugar, however, retards the formation of glucose-1-phosphate from the cyclic compound by the enzymes of the cell extract: In the presence of glucose the amylomaltase is incapable of synthesizing substrates for the phosphorylase from maltose. This experimental result clearly demonstrates that the amylomaltase is involved in the disproportionation of maltosaccharides arising from the cyclodextrins.
3. A NADP⁺-specific glucose dehydrogenase was demonstrated to be present in the cell extracts. This enzyme, which is activated by ADP, may control the energy-depending pool of free glucose. Glucose originates from the disproportionation of maltosaccharides catalyzed by the glucanotransferases.
4. A glucose-1-phosphate-hydrolysing phosphatase, which is shown to be present in the cell extract, seems to be without physiological significance for the metabolism of the cyclodextrins.
5. Preliminary permeation studies make it probable that the cyclodextrins are transported into the cells as such and degraded only within the cells.
6. A scheme for the metabolism of cyclodextrins in Klebsiella pneumoniae M 5 al is proposed.

     

Cyclodextrin-Glucanotransferase von Klebsiella pneumoniae

1. The strain M 5 al of Klebsiella pneumoniae grows excellently with starches. We were able to show that besides the pullulanase associated with the external membrane of the cells the bacterium produces an inducible, extracellular cyclodextrin glucanotransferase [1,4-alpha-D-glucan-4-alpha-(1,4-alpha-glucano)-transferase (cyclising) (EC 2.4.1.19)]. Potato starch and cyclohexaamylose or cycloheptaamylose were found to be the best "inducing" carbon sources for the synthesis of the enzyme. When the bacteria are grown batchwise, maltose is a poorly "inducing" carbon source; larger quantities of the enzyme are synthesized by continuous cultivation with maltose as growth limiting factor. 2. For the determination of the cyclodextrin glucanotransferase-activity an assay method wsa worked out. 3. The enzyme could be separated from the culture filtrate and purified to more than 90% in few steps. At a total yield of 61.2% related to the activity of the culture filtrate employed we received an enzyme solution with the specific activity of 26.6 units/mg protein. Some properties of the enzyme are described. 4. The products formed from amylopectin by the enzyme were analyzed. Somewhat more than half the amylopectin was found as cyclodextrins. 29.3% of the cyclodextrin fraction were cycloheptaamylose, 47.2% cyclohexaamylose and 10.7% exo-branched cyclohexaamylose. 12.8% of cyclohexaamylose were obtained from a cyclodextrin glucanotransferase-limit dextrin after debranching by pullulanase and exposing the product to the action of the glucanotransferase again. 5. The importance of the cyclodextrin glucanotransferase for the utilization of starches by this strain of Klebsiella pneumoniae is discussed. After a first characterization the enzyme is compared to the amylase of Bacillus macerans.     

绿原酸联合左氧氟沙星对肺炎克雷伯菌生物膜的体外抑制现象观察

目的观察绿原酸联合左氧氟沙星对肺炎克雷伯菌生物膜的体外抑制现象。方法收集广州市中医医院临床分离的肺炎克雷伯菌株(排除同一患者同一部位重复菌株),应用半定量结晶紫法进行生物膜形成能力测定;选取3株生物膜强阳性的菌株,采用微量肉汤稀释法,对绿原酸、左氧氟沙星用MH肉汤倍比稀释,测定两者的最低抑菌浓度;棋盘稀释法测定绿原酸与左氧氟沙星的协同抑菌浓度;建立体外生物膜模型,经药物干扰,观察药物对生物膜形态的影响。结果在62株菌中共有33株为生物膜阳性,占53.2%,绿原酸对实验菌株的MIC分别是2 048、2 048和2 048μg/mL,左氧氟沙星的MIC分别是32、64和32μg/mL,两药联合作用,部分抑菌浓度指数(FIC)是0.5、0.5和0.5,提示两药呈协同作用,经体外建模及药物干扰,镜下药物组的黑色絮状物较未处理组明显减少。结论绿原酸具有抑制肺炎克雷伯菌生物膜作用,与左氧氟沙星联合,作用效果更显著。     

Protective activity of a cell-free Klebsiella vaccine in relation to different Klebsiella pneumoniae serovars

The capacity of dried Klebsiella cell-free vaccine, obtained from strain No. 204 by the disintegration of microbial cells with hydroxylamine, for protecting mice from Klebsiella septic infection caused by the homologous serovar and 9 heterologous serovars of K. pneumoniae was studied. The newly developed preparation was found capable of stimulating immunity not only to the homologous K. pneumoniae serovar, but also to other K. pneumoniae heterologous serovars: K1, K9, K11, K16, K20, K61. The protective capacity of the preparation with respect to these serovars was not inferior to that of the vaccines prepared by the same method from the corresponding homologous strains. The capacity of the vaccine to protect mice from Klebsiella sepsis was manifested irrespective of the virulence of the strains used for challenge.