Hemagglutinins of Klebsiella pneumoniae and K. oxytoca isolated from different sources.

482 Klebsiella pneumoniae and K. oxytoca strains isolated from different sources (human clinical material, feces of healthy subjects, sewage) were investigated for expression of MS-(mannose-sensitive) and MR/K-(mannose-resistant, Klebsiella-like) hemagglutinins. Regardless of the source of isolation, the majority of K. pneumoniae strains induced mannose-sensitive hemagglutination, but very few K. oxytoca isolates produced this type of hemagglutinin. In both species, most strains induced MR/K-hemagglutination. In general, environmental isolates showed a lesser incidence in production of each hemagglutinin and a higher percentage of non-hemagglutinating strains than fecal and clinical strains. These results suggest clinical isolates to be phenotypically more related to fecal than to environmental strains.

A reverse approach to 1H-n.m.r. assignments of bacterial polysaccharides.

A new approach is reported for obtaining the assignment of the 1H-n.m.r. spectra of bacterial polysaccharides which are not amenable to analysis using conventional strategies. An unambiguous assignment of the 13C-n.m.r. spectrum is made by 13C-COSY of the polysaccharide labelled to a high level with 13C. Assignment of the 1H-n.m.r. spectrum is then made by proton-detected CH-correlation spectroscopy of material that is labelled at a low level. Using this approach, virtually complete assignments have been made for the Klebsiella K3 serotype polysaccharide under physiological conditions of temperature and pH.

Glycosidase activities in alveolar macrophages from guinea pigs stimulated with a glyco-proteic complex extracted from Klebsiella pneumoniae.

An increased resistance of laboratory animals to pulmonary infections following per os administration of a glyco-proteic complex extracted from Klebsiella pneumoniae has been reported. This was associated with an increased phagocytic capacity of alveolar macrophages (AM). In this report, the effect of treating guinea pigs with this extract on the alveolar macrophage (AM) glycosidase machinery has been studied. AM were collected by bronchoalveolar lavage, the cells were pelleted by centrifugation and AM were purified by adherence on plastic dishes. Sialidase, beta-galactosidase, beta-glucuronidase and N-acetyl-beta-D glucosaminidase activities were measured in the AM homogenate. In order to evaluate an extracellular release of these enzymes, they were also assayed in the cell free lavage fluid. Lactic dehydrogenase (LDH) activity was assayed as a control for cell lysis. In treated animals, the total number of cells as well as the number of AM increased by 25% (ns). The protein concentration was slightly reduced in the cell homogenate and unchanged in the lavage fluid. The only significant change was a decreased sialidase activity, in AM homogenate (p less than or equal to 0.01) and in lavage fluids (ns). The LDH activity was not increased in the lavage fluids.

Comparison of SDS-PAGE protein patterns with other typing methods for investigating the epidemiology of 'Klebsiella aerogenes'.

Twenty-four cultures comprising 20 clinical isolates of 'Klebsiella aerogenes' from two hospitals, a reference strain of 'K. aerogenes' and the type strains of three other Klebsiella species, were characterized by one-dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) of whole-cell proteins. The protein patterns were highly reproducible and were used as the basis of a numerical analysis which divided the clinical isolates into 12 protein types. Comparison with established typing methods indicated that the level of discrimination of SDS-PAGE was similar to that achieved with conventional typing methods but the strains were grouped differently. Protein typing subdivided five serotype K3 isolates that could also be distinguished by phage typing. Conversely, three strains of protein type 11 were clearly distinguishable by both serotyping and phage typing. We conclude that high-resolution SDS-PAGE of proteins provides an effective adjunct to other methods for typing isolates of 'K. aerogenes'.

Solubilization and reconstitution of the Na(+)-dependent citrate carrier of Klebsiella pneumoniae.

The citrate carrier of Klebsiella pneumoniae fermenting this substrate has been solubilized from the bacterial membranes with Triton X-100. The transport function was reconstituted by incorporation of the carrier into proteoliposomes using a freeze-thaw sonication procedure. Citrate uptake into these proteoliposomes required the presence of Na+ ions on the outside; the amount of citrate accumulated increased as the external Na+ concentration increased from 0 to 100 mM. Proteoliposomes preloaded with citrate catalyzed citrate counterflow when added to external [14C] citrate. Sodium ions were required for counterflow activity. The kinetics of citrate uptake, counterflow, or efflux were not influenced by an inside negative membrane potential, and the presence of the uncoupler carbonyl cyanide p-trifluoromethoxyphenylhydrazone was without effect on citrate uptake. The data therefore suggest an electroneutral Na(+)-citrate symport mechanism for the transport of this tricarboxylic acid into K. pneumoniae.

Interaction of Mg2+ and Ca2+ in in vitro hexagonal assembly of R-form lipopolysaccharides.

The R-form lipopolysaccharide (LPS) from Klebsiella pneumoniae strain LEN-111 (03-:Kl-), from which cationic material had been removed by electrodialysis, formed an orderly hexagonal lattice structure when suspended in 50 mM Tris buffer at pH 8.5 containing MgCl2. The center-to-center distance (lattice constant) of the hexagonal lattice structure depended upon the concentration of MgCl2 and reached the shortest value (15 nm) at 10 mM. In contrast, CaCl2 could not produce the orderly hexagonal lattice structure but produced an irregular network structure with a center to center distance of 19 to 20 nm. When the LPS was suspended in Tris buffer containing 10 mM MgCl2 mixed with 1 or 10 mM CaCl2, formation of the orderly hexagonal lattice structure of the magnesium salt type was inhibited and the LPS showed the structure of the calcium salt type. When 1 or 10 mM CaCl2 was mixed with 10 mM MgCl2, the binding of Mg to the LPS was significantly inhibited compared with when 10 mM MgCl2 was added alone. On the contrary, when 10 mM CaCl2 was mixed with 10 mM MgCl2, the binding of Ca to the LPS was enhanced compared with when 10 mM CaCl2 was added alone. It was therefore concluded that the inhibition of formation of the hexagonal lattice structure of the magnesium salt type by addition of CaCl2 is due to the inhibition of the binding of Mg to the LPS. Such a competitive interaction of Mg2+ and Ca2+ was also observed with the electrodialyzed LPS of Escherichia coli K-12.

Characterisation of the Klebsiella pneumoniae nitrogen-fixation regulatory proteins NIFA and NIFL in vitro.

Transcriptional activation by the Klebsiella pneumoniae nitrogen-fixation-specific positive control protein, NIFA, (nifA gene product) has been demonstrated in vitro in S30 extracts from cells which overproduce this protein. The activity of NIFA was dramatically reduced in vitro in the presence of the negative regulatory protein NIFL (nifL gene product) but was not inhibited by the presence of a mutant NIFL protein, NIFL2404. Transcriptional activation from the nifH promoter by NIFA was dependent on the alternative sigma factor, sigma 54, and also required the presence of an upstream activator sequence. NIFA activity was temperature-sensitive in vitro (as it is in vivo) which is due, at least in part, to the intrinsic lability of the protein itself. The majority of overproduced NIFA and NIFL was insoluble after low-speed centrifugation and was inactive in vitro. A low level of less aggregated NIFA protein present in cell extracts was responsible for in vitro activity and this fraction was partially purified.

Relationship of high tissue concentrations of azithromycin to bactericidal activity and efficacy in vivo.

Measurement of killing kinetics of azithromycin against strains of Streptococcus pneumoniae and Klebsiella pneumoniae in vitro showed that it had a limited bactericidal activity (greater than 90% kill) for the first eight hours of incubation, but developed complete bactericidal activity (greater than 99.9% kill) by 24 h incubation. Since high and sustained tissue levels of azithromycin occur in animals and humans, it was proposed that it might produce a bactericidal effect in vivo. This was demonstrated in a lung infection model in mice, designed to mimic the in-vitro killing studies. A 25 mg/kg dose of azithromycin given 24 h before intranasal challenge reduced the recoverable Str. pneumoniae population by greater than 99.9%, in comparison with untreated controls. Erythromycin did not produce a bactericidal effect at 100 mg/kg, and roxithromycin only reduced the viable count by 96%, at a dose of 50 mg/kg. Against a K. pneumoniae lung infection, a 50 mg/kg dose of azithromycin reduced the bacterial count by 99%. The bactericidal effect was correlated with lung tissue concentrations of azithromycin. In a proliferating Escherichia coli paper disc infection model, extravascular fluid concentrations of azithromycin were correlated with a 99.9% reduction in bacterial count, while corresponding serum concentrations were always less than the MIC. Dosing with azithromycin eradicated Haemophilus influenzae from the bulla (middle ear) of gerbils, as was not the case with erythromycin and roxithromycin. This effect was correlated with the antibiotic concentration in bulla lavage.

Comparative effects of F1 and P1 fractions obtained from a Klebsiella pneumoniae glycoproteic extract (RU 41740) on polymorphonuclear leukocytes.

RU 41740 is a glycoprotein extract from Klebsiella pneumoniae described as a macromolecular aggregation of a lipopolysaccharide (LPS)-associated protein (F1 fraction) and a glycoproteic complex (P1 fraction). The human polymorphonuclear (PMN) response was studied after incubation of the cells in the presence of RU 41740, F1 and P1 fractions, or F1-P1 complex. Oxidative metabolism was assessed by chemiluminescence, O2 consumption, O2- generation, and degranulation by beta-glucuronidase release. Results were compared to data obtained with a homologous LPS. RU 41740, F1 fraction, and F1-P1 complex increased the respiratory burst of PMNs stimulated by opsonized zymosan (OZ). N-formylmethionylleucylphenylalanine (fMLP), phorbol myristate acetate, or the calcium ionophore A23187. The beta-glucuronidase release was stimulated by the same compounds when OZ or fMLP were used as stimuli. These effects were dose-dependent. In contrast, P1 fraction was inactive. Addition of polymyxin B resulted in a profound inhibition of both the F1 fraction and LPS activities but only in a partial inhibition of RU 41740 effects. These results strengthen the hypothesis that different biochemical pathways are involved in the enhancement of stimulated neutrophil functions by RU 41740.

[The compositional characteristics of the cellular fatty acids in strains of Klebsiella pneumoniae serovar K 1]. / Osobennosti sostava kletochnykh zhirnykh kislot shtammov Klebsiella pneumoniae serovara K 1.

Klebsiella pneumoniae, serovar K1 strains, differ essentially in the composition of cellular fatty acids from serovar strains K 2, K 3, K 7-K 72, K 74-K 80 of this species and are analogous to K. rhinoscleromatis and K. ozaenae by the given trait. The differences concern the concentration of cyclopropane fatty acids and their biosynthetic precursors--monounsaturated fatty acids, that is typical of a number of other bacteria belonging to the related species and, evidently, may be a marker of phenetic differences at the level of related taxonomic groups. The data obtained evidence for taxonomic separation of K. pneumoniae serovar K1 strains from representatives of other serovars of the given species.

The Q-linker: a class of interdomain sequences found in bacterial multidomain regulatory proteins.

Evidence is presented that establishes a novel class of interdomain linkers, named Q-linkers, as a defined element of protein structure. Q-linkers occur at the boundaries of functionally distinct domains in a widespread set of bacterial regulatory and sensory transduction proteins, typified by the nitrogen regulatory proteins, NtrB, NtrC, NifA and NifL. Q-linkers are not strongly conserved in sequence in otherwise homologous proteins, are approximately 15-25 residues long and relatively rich in glutamine, arginine, glutamate, serine and proline, and are assigned as 'coil', with a very low probability of alpha or beta structure, by eight secondary structure prediction methods. Hydrophobic amino acids are spaced with a periodicity of approximately 4-5 residues in the C-terminal 15 residues of these sequences. A pattern discriminator is presented that incorporates these properties and is used to predict segments resembling Q-linkers in sequence databases. Insertions of four and eight amino acids, constructed in the Q-linker sequences of NtrC and NifA, were found to have no effect on the function of the proteins in signal transduction and transcriptional activation. However, when NtrC was expressed as two separate polypeptides, consisting of the domains normally joined by the Q-linker, the construct failed to function. These results suggest that the Q-linker serves a simple but essential role in tethering the structurally-distinct but interacting domains of the protein. Q-linkers are therefore potentially applicable as domain fusion junctions for engineered chimaeric multidomain proteins expressed in enteric bacterial systems.

Two-dimensional gas chromatography with electron-capture detection used in the determination of specific peptidoglycan and lipopolysaccharide constituents of gram-negative bacteria in infected human urine.

Gas chromatography with electron-capture detection was used in the determination of diaminopimelic acid (as the N-heptafluorobutyryl isobutyl derivative) and 3-hydroxymyristic acid (as the O-pentafluorobenzoyl methyl derivative) in Gram-negative bacterial cells in infected human urine. Use of the column-switching (two-dimensional gas chromatography) technique greatly enhanced the selectivity of the detection and simplified the processing of samples. The system described should prove useful for trace detection of specific bacterial constituents in complex environments.

Comparison of multidimensional scaling and principal component analysis of interspecific variation in bacteria.

Multidimensional scaling (MDS) and principal component analysis (PCA) were applied to bacterial taxonomy. The biochemical profiles of 42 isolates consisting of four species of Enterobacteriaceae were used. Both MDS and PCA use proximity measures such as the correlation coefficient or Euclidean distance to generate a spatial configuration (map) of points in multidimensional space where distances between points reflect the similarity among isolates. Multidimensional scaling and principal component analysis were able to discriminate organisms in two dimensions. The test components of the MDS and PCA factors (derived variables composed of linear combination of biochemical tests) were different for a two-dimensional solution.

Molecular cloning and purification of klebicin B.

A novel klebicin, klebicin B, produced by an isolate of Klebsiella pneumoniae has been identified. It is encoded by a 5.5 kb plasmid, pKlebB-K17/80, which is mobilized into K. pneumoniae UNF5023 by a large plasmid found in the same strain. The 5.5 kb plasmid has been cloned into the high-copy-number vector pUC19 and the restriction map of the resulting recombinant plasmid pRJ180 has been determined. Using sub-cloning and transposon mutagenesis, the klebicin B structural gene, the klebicin B immunity gene and the mitomycin C (MC) sensitivity gene (lys) present on pRJ180 have been localized. Transposon inserts which inactivated klebicin production also abolished lysis protein production encoded by pRJ180, but did not affect klebicin B immunity. Using SDS-PAGE an MC-induced polypeptide of 85 kDa was observed in cultures of K. pneumoniae UNF5023(pRJ180). This polypeptide was absent in cultures carrying plasmid pRJ180 with a Tn1000 insert which inactivated klebicin production. Analysis of the polypeptides present in the medium of Escherichia coli JM83 hsdR(pRJ180) or K. pneumoniae UNF5023(pRJ180) indicated that the 85 kDa polypeptide is specifically secreted from the producing cell. Klebicin B has been purified, using gel filtration, from a cell-free extract of K. pneumoniae UNF5023(pRJ180) which had been induced with MC. After boiling in sample buffer the purified klebicin B gave rise to two peptides on SDS-PAGE, one of 85 kDa and the other of 11 kDa. Klebicin B-resistant mutants of K. pneumoniae UNF5023 were sensitive to klebicin A, colicin B and colicin D.

Formation of a hexagonal lattice structure by an R-form lipopolysaccharide of Klebsiella: effect of various divalent cations on the lattice formation.

The R-form lipopolysaccharide from Klebsiella pneumoniae strain LEN-111 (O3-:K1-), from which cationic material had been removed by electrodialysis, was previously shown to form a hexagonal lattice structure with the lattice constant of 14 to 15 nm when suspended in 50 mM tris(hydroxymethyl)aminomethane buffer at pH 8.5 containing 10 mM Mg2+. Under this experimental condition, effects of other divalent metal cations on the hexagonal assembly of the electrodialyzed LPS were compared with that of Mg2+. The Zn2+, Hg2+, Cu2+, and Ni2+ could produce essentially the same hexagonal lattice structure with the lattice constant of 14.5 to 15.0 nm as that formed with Mg2+. The Cd2+, Co2+, and Fe2+ produced the hexagonal lattice structure with the lattice constant of 15.5 to 16.0 nm, and Ba2+, Sr2+, and Ca2+ produced that with the lattice constant of 18 to 19 nm. In addition, the hexagonal lattice structures formed with the latter three cations were less orderly than those formed with the other cations. When the higher concentrations of Ba2+, Sr2+, and Ca2+ were used, the lattice constants were not shortened. The length of lattice constants of the hexagonal lattice structures formed with the divalent cations did not relate to the quantity of the cations bound to the LPS. Among the divalent cations tested, Hg2+ was bound to the LPS in the smallest amount (its atomic ratio to P, 0.07), and Zn2+ and Fe2+ were bound in very large amounts (their atomic ratios to P, 2.94 and 8.28, respectively).

Purification of the lipopolysaccharide fraction from Klebsiella pneumoniae O1 K2 by high-performance liquid chromatography.

An high-performance liquid chromatography technique was applied to purify the lipopolysaccharide fraction from a lysate of Klebsiella pneumoniae O1 K2 (NCTC 5055). The separation of the lipopolysaccharide fraction from the proteins was carried out with a reversed-phase column. By this method the lipopolysaccharide fraction was obtained in a pure state, devoid of proteins but possessing the same biological properties as the lipopolysaccharide fraction prepared by the classical phenol-water technique.

Molecular weight determination and partial characterization of Klebsiella pneumoniae hemolysins.

Two thiol-activated Klebsiella pneumoniae hemolysins were purified from growth media by means of salt precipitation, gel filtration, ion-exchange chromatography, and polyacrylamide gel electrophoresis. The hemolysins peaks coincided with the protein and glycoprotein peaks as determined by chromatography and electrophoresis. The molecular weights, estimated by gel filtration, were 8400 and 19,000; by sodium dodecyl sulfate--polyacrylamide gel electrophoresis, the values were calculated as 15,500 and 27,000. The electrophoretic bands were best detected by the periodic acid--Schiff method. Reduction of the disulfide linkages did not cause the originally larger molecule to break into 8400 and 19,000 hemolysins. However, trypsin treatment cleaved the 19,000 hemolysin into an active moiety, with an electrophoretic migration similar to the 8400 hemolysin. A naturally occurring proteolytic activity was investigated using pepstatin and antipain. When the trypsin inhibitor was added to the system, the hemolytic activity was detected only in the 19,000 hemolysin and the smaller hemolysin was absent.

Characterization of pathogenic bacteria by automated headspace concentration-gas chromatography.

Automated headspace concentration-gas chromatography (AHC-GC) was used to profile the volatile metabolites produced by Proteus mirabilis, Klebsiella pneumoniae, Pseudomonas aeruginosa and Staphylococcus aureus. Bacterial cultures were incubated in trypticase soy broth and examined at 24 h. The profiles were consistent for each genus examined and variation observed among the different strains of each species was chiefly quantitative. The volatiles were identified by concurrent headspace concentration-gas chromatography-mass spectrometry and consisted mainly of isobutanol, isopentanol, isopentyl acetate, 1-undecene and methyl ketones. There were sufficient differences in the profiles in the 4-6 min elution period to distinguish P. aeruginosa and S. aureus from each other and from the other two bacteria. P. mirabilis and K. pneumoniae typically showed three intense peaks which corresponded to isobutanol, isopentyl acetate and isopentanol. The determination of volatiles by AHC-GC is sensitive, rapid and offers a possible alternative for automatic detection and characterization of pathogenic bacteria.

Purification and characterization of a bacteriocin from Klebsiella pneumoniae 158.

Klebocin, a bacteriocin produced by Klebsiella pneumoniae 158, was purified to homogeneity by ammonium sulphate fractionation and sequential DEAE-Sephacel and Sephadex G-150 column chromatography. The purified preparation had an Mr of approximately 40 000 on SDS-PAGE. Chemical analysis of the purified preparation showed it to be a protein, and it was sensitive to digestion by various proteolytic enzymes.