Abiotic process for Fe(II) oxidation and green rust mineralization driven by a heterotrophic nitrate reducing bacteria (Klebsiella mobilis).

Green rusts (GRs) are mixed Fe(II)-Fe(III) hydroxides with a high reactivity toward organic and inorganic pollutants. GRs can be produced from ferric reducing or ferrous oxidizing bacterial activities. In this study, we investigated the capability of Klebsiella mobilis to produce iron minerals in the presence of nitrate and ferrous iron. This bacterium is well-known to reduce nitrate using an organic carbon source as electron donor but is unable to enzymatically oxidize Fe(II) species. During incubation, GR formation occurred as a secondary iron mineral precipitating on cell surfaces, resulting from Fe(II) oxidation by nitrite produced via bacterial respiration of nitrate. For the first time, we demonstrate GR formation by indirect microbial oxidation of Fe(II) (i.e., a combination of biotic/abiotic processes). These results therefore suggest that nitrate-reducing bacteria can potentially contribute to the formation of GR in natural environments. In addition, the chemical reduction of nitrite to ammonium by GR is observed, which gradually turns the GR into the end-product goethite. The nitrogen mass-balance clearly demonstrates that the total amount of ammonium produced corresponds to the quantity of bioreduced nitrate. These findings demonstrate how the activity of nitrate-reducing bacteria in ferrous environments may provide a direct link between the biogeochemical cycles of nitrogen and iron.

Bacterial succession and degradative changes by biofilm on plastic medium for wastewater treatment.

Biofilms contain a diverse range of microorganisms and their varying extracellular polysaccharides. The present study has revealed biofilm succession associated with degradative effects on plastic (polypropylene) and contaminants in sludge. The wet weight of biofilm significantly (p < 0.05) increased; from 0.23 ± 0.01 to 0.44 ± 0.01 g. Similarly, the dry weight of the biofilm increased from 0.02 to 0.05 g. Significant reduction in pathogens (E. coli and feacal coliforms) by MPN technique (>80%) and in chemical parameters (decrease in COD, BOD5 of 73.32 and 69.94%) representing diminution of organic pollutants. Energy dispersive X-ray spectroscopy (EDS) of plastic revealed carbon and oxygen contents, further surface analysis of plastic by scanning electron microscopy (SEM) revealed emergence of profound bacterial growth on the surface. Fourier transform infrared (FTIR) spectroscopy conforms its biotransformation under aerobic conditions after 8 weeks. New peaks developed at the region 1050 and 969 cm(-1) indicating CO and CC bond formation. Thus plastic with 6 weeks old aerobic biofilm (free of pathogens, max. weight, and OD, efficient COD & BOD removal ability) is suggested to be maintained in fixed biofilm reactors for wastewater treatment.

A new mathematical model to evaluate simazine removal in three different immobilized-biomass reactors.

A new mathematical model based on the cinetical Langmuir equation is developed to interpret and predict the effectiveness of simazine (SZ) removal in immobilized-biomass reactor (IBR), to consider herbicide-support affinity (Cx), and herbicide-cell affinity (Cy). Three solid supports: sepiolite monolith, granular sepiolite, and alginate were used in pilot-scale reactors that were inoculated with Klebsiella planticola DSZ. The abiotic process was analysed by measuring the SZ sorption capacity of the reactor supports. Sepiolite monolith showed the maximum value for herbicide-support affinity (28.02+/-0.9%). The effectiveness of the biotic process was estimated considering the formation of biomass and SZ biodegradation. Granular sepiolite showed either higher affinity with SZ and viability rate (0.90) throughout the process, and SZ removal rate was 3.39+/-0.06 mg/h. The mathematical model presented in this paper provides useful insights into the interpretation of experimental data as well as prediction for the implementation of biological reactors.

Klebsiella planticola strain DSZ mineralizes simazine: physiological adaptations involved in the process.

We examined the ability of a soil bacterium, Klebsiella planticola strain DSZ, to degrade the herbicide simazine (SZ). Strain DSZ is metabolically diverse and grows on a wide range of s-triazine and aromatic compounds. DSZ cells grown in liquid medium with SZ (in 10 mM ethanol) as carbon source mineralized 71.6+/-1.3% of 0.025 mM SZ with a yield of 4.6+/-0.3 microg cell dry weight mmol(-1) carbon. The metabolites produced by DSZ during SZ degradation included ammeline, cyanuric acid, N-formylurea and urea. We studied the physiological adaptations which allow strain DSZ to metabolize SZ. Using scanning electron microscopy, we detected DSZ cells covering the surfaces of SZ crystals when the herbicide was used at high concentrations (0.1 mM). The membrane order observed by FTIR spectroscopy showed membrane activity at low temperature (4 degrees C) to assimilate the herbicide. Membrane fatty acid analysis demonstrated that strain DSZ adapted to grow on SZ by increasing the degree of saturation of membrane lipid fatty acid; and the opposite effect was detected when both SZ and ethanol were used as carbon sources. This confirms the modulator effect of ethanol on membrane fluidity.

Comparison of atomic force microscopy interaction forces between bacteria and silicon nitride substrata for three commonly used immobilization methods.

Atomic force microscopy (AFM) has emerged as a powerful technique for mapping the surface morphology of biological specimens, including bacterial cells. Besides creating topographic images, AFM enables us to probe both physicochemical and mechanical properties of bacterial cell surfaces on a nanometer scale. For AFM, bacterial cells need to be firmly anchored to a substratum surface in order to withstand the friction forces from the silicon nitride tip. Different strategies for the immobilization of bacteria have been described in the literature. This paper compares AFM interaction forces obtained between Klebsiella terrigena and silicon nitride for three commonly used immobilization methods, i.e., mechanical trapping of bacteria in membrane filters, physical adsorption of negatively charged bacteria to a positively charged surface, and glutaraldehyde fixation of bacteria to the tip of the microscope. We have shown that different sample preparation techniques give rise to dissimilar interaction forces. Indeed, the physical adsorption of bacterial cells on modified substrata may promote structural rearrangements in bacterial cell surface structures, while glutaraldehyde treatment was shown to induce physicochemical and mechanical changes on bacterial cell surface properties. In general, mechanical trapping of single bacterial cells in filters appears to be the most reliable method for immobilization.

The survival and growth of an environmental Klebsiella isolate in detergent solutions.

A Klebsiella isolate (G1) was capable of survival and growth in an environment containing high levels of anionic (15.6%) and non-ionic detergent (7.8%). Cell numbers were monitored by traditional viable plate counts (culturability), and by staining with Rhodamine 123 (vital counts) and Acridine Orange (total counts). On inoculation into detergent solutions, only 10% of cells retained vitality (rhodamine 123) after 24 h. Of those, only 1% were able to form colonies on artificial culture media. Under low nutrient conditions (TOC < 0.89 mg l - 1), no recovery was observed over a 96 h period, but addition of nutrients (TOC 3.84 mg l - 1) allowed recovery within 48 h. Such cells were initially (24 h) elongated, but subsequently (144 h) returned to their normal size. In the presence of detergent without added nutrient, cell size was reduced after 144 h. After adaptation to the detergent environment (24 - 48 h), cell numbers in detergent with added nutrient broth (vital, viable and total) rose, until levels equivalent to those of a detergent-free control were attained. Detergent solutions provide a stressful environment. Nutrient levels in the detergent solutions affected cell size and the ability of the Klebsiella (G1) to survive and grow.

Formate and ethanol are the major products of glycerol fermentation produced by a Klebsiella planticola strain isolated from red deer.

The rumen contents of red deer (Cervus elaphus) were used to isolate bacterial capable of fermenting glycerol. The biochemistry, physiology, morphology and phylogeny of one isolate were studied in detail. The isolate (DR3) was tentatively identified as a strain of the species Klebsiella planticola as based on phenotypic characterization. The data obtained from 16S rRNA sequence analysis showed that the deer rumen isolate DR3 was 99.7% similar to the type strain of Kl. planticola (DSM 3069T), thus confirming the results of the phenotypic characterization. During cell growth, it was established that glycerol dissimilation by Kl. planticola DR3 led to the production of formate and ethanol at equimolar levels of 32 mmol 1(-1) and 30 mmol 1(-1), respectively. As a result of the data obtained, a closed carbon balance was constructed for Kl. planticola DR3. This finding represented the first report of the complete end-product profile for glycerol dissimilation by a strain of Kl. planticola isolated from cervine rumen contents.

Differentiation of viable and non-viable bacterial biofilms using electrorotation.

A new technique for studying the properties of biofilms has been developed, based on the phenomenon of electrorotation. Biofilms of Klebsiella rubiacearum were formed on the surfaces of 6 microns diameter polystyrene beads, and the presence of such films was found to alter their electrorotation spectra. The effects of adding a biocide (polyhexanide) to the surrounding aqueous medium was also investigated. The dielectric properties of the beads with biofilms, before and after biocide treatment, were interpreted from the electrorotation spectra using modelling methods that have been well tested for other heterogeneous biological systems. The technique is of value in understanding the physico-chemical properties of biofilms and can be adapted for monitoring the presence of toxic chemicals and for testing the activity of biocides against biofilms.

[The surface structures and biological properties of Klebsiella strains]. / Poverkhnostnye struktury i biologicheskie svoistva shtammov klebsiell.

The electron-microscopic method has been used to study the surface structures and biological properties (adhesiveness, antibiotic-resistance, biochemical properties) in 67 strains of Klebsiella isolated from one-year-old children with intestinal infections (56) and from healthy ones (11). It is found that bacterial cells in 58.2% of strains have fimbriae of the 3d type, in 20.9%--of the 1st type. No correlation has been revealed between the type of fimbriae and the studied properties.

Adhesion of fimbriated and non-fimbriated Klebsiella strains to synthetic polymers.

Adherence of three fimbriated and non-fimbriated Klebsiella strains to polyetherurethane was investigated in order to assess the possible role of fimbriae in the adhesion of Klebsiella to synthetic polymers. Fimbriated strains with type 1 and type 1.3 fimbriae adhere significantly stronger to polyurethane than the same strains lacking fimbriae, whereas a strain with type 3 fimbriae shows no different adherence compared with the non-fimbriated variant. Analysis of adhesion kinetics and isotherms reveals that adherence of fimbriated Klebsiella strains is proceeded by the formation of multicellular bacterial layers on the polymer surface. Measurements of the relative hydrophobicity of the strains and adherence experiments under exclusion of unspecific interactions point out that fimbriae obviously play a more important role in adhesion than relative hydrophobicity. The demonstration of reduction of bacterial adherence to polyetherurethane by blocking fimbrial action with fimbriae-specific sugars supports this further.

Chemiluminescence response of human polymorphonuclear leukocytes induced by purified, latex attached Klebsiella fimbriae.

Type 1 fimbriae (T1F) and type 3 fimbriae (T3F) were isolated from Klebsiella species, purified, attached to latex beads and tested for their ability to stimulate human polymorphonuclear leukocyte (PMNL) oxidative activity. The luminol dependent chemiluminescence assay was used to evaluate the response of phagocytes. Latex particles coated with type 3 fimbriae (1-T3F) induced a significantly higher chemiluminescence response than those with type 1 fimbriae (1-T1F). Opsonization of 1-T1F with pooled human serum induced chemiluminescence responses which were statistically significantly enhanced as compared to opsonized 1-T3F and both kinds of non-opsonized fimbriae.

[The hemagglutinating activity of bacteria in the genus Klebsiella and the morphofunctional characteristics of their fimbriae]. / Gemaggliutiniruiushchaia aktivnost' bakterii roda Klebsiella i morfofunktsional'nye osobennosti ikh fimbrii.

The hemagglutinating activity of 77 Klebsiella strains from the international collection, grown in a culture medium prepared on the basis of soy-bean flour enzymatic hydrolysate, was studied. These strains could be divided into four groups according to their capacity for synthesizing different types of hemagglutinins on their surface: 2 strains carried mannose-sensitive hemagglutinins, 18 strains had mannose-resistant K-type hemagglutinins, 48 strains exhibited the signs indicating the presence of both mannose-sensitive and mannose-resistant hemagglutinins, and 9 strains showed no hemagglutinating activity. The hemagglutinating activity of strains K-74, K-79, K-80, K-81 and K-82 was characterized. Of the reference strains under study, 22 strains were found to have mannose-resistant hemagglutinating activity with respect to fresh chick red blood cells. The occurrence of hemagglutinins in Klebsiella was shown to depend on the temperature of cultivation and the consistency of the culture medium. The formation of large-sized capsules in Klebsiella grown in the Werfel-Fergusson medium with a considerable content of saccharose was shown to cause the absorption of their fimbrial structures by the capsular substance and, as a consequence, the suppression of their hemagglutinating activity.

Effect of sub-minimal inhibitory concentrations of ciprofloxacin and fleroxacin on the bacterial capsular antigen and opsonophagocytosis by human polymorphonuclear leukocytes.

We have studied the effect of sub-minimal inhibitory concentrations (sub-MIC) of ciprofloxacin and fleroxacin on capsulated (K+) and non-capsulated (K-) Gram-negative bacilli (Escherichia coli O1:K1, O7:K1, O1:K-, O7:K-, and Klebsiella oxytoca) as well as on Staphylococcus aureus and we investigated the interaction of antibiotic pretreated bacteria with human serum and polymorphonuclear leukocytes (PMN). Following overnight growth in the presence of 1/2 MIC of the antibiotics, bacteria were opsonized in human serum and incubated with PMN. Opsonophagocytosis was quantified as the ratio of uptake by PMN of radioactively labeled bacteria. Ciprofloxacin and fleroxacin enhanced the phagocytosis rate of E. coli K+ strains (control 5-10%; 1/2 MIC of ciprofloxacin and fleroxacin 70-80%) of K. oxytoca (control O-2%; 1/2 MIC of ciprofloxacin and fleroxacin 35-40%) as well as of Staph. aureus (control 5-10%; 1/2 MIC of the antibiotics 35-40%). Opsonophagocytosis of K- strains was not altered. The enhancement of opsonophagocytosis was a complement dependent process. Exposure of capsulated E. coli to ciprofloxacin as well as to fleroxacin resulted in decreased amounts of the capsular antigen.

Type of fimbriation determines adherence of Klebsiella bacteria to human epithelial cells.

Clinical isolates of three Klebsiella strains (encapsulated and nonencapsulated mutants) with type 1 (mannose-sensitive, MS+MR-), type 3 (mannose-resistant, MS-MR+) and type 1 and 3 (MS+MR+) fimbriae were investigated for their ability to adhere to epithelial cells. Considerable adhesion to human buccal, tracheal, pulmonary and uroepithelial cells could be demonstrated. Independent of encapsulation and type of epithelial cells, adhesion of MS+MR+ (type 1.3) fimbriated Klebsiella bacteria was significantly stronger than adhesion of microorganisms carrying only type 1 (MS+MR-) or type 3 (MS-MR+) fimbriate, respectively. Adherence of nonencapsulated type 1 and 3 (MS+MR+) fimbriated Klebsiella bacteria to mammalian cells was significantly inhibited in the presence of D-mannose. Certain carbohydrates (D-glucose, D-galactose) did not interfere with this adhesion process.

[Ultrastructural studies of Klebsiella rhinoscleromatis]. / Ul'trastrukturnye issledovaniia Klebsiella rhinoscleromatis.

In 1870, F. Hebra described a slowly progressing granulomatous disease of the nasal mucous membrane he called rhinoscleroma. Later, N. M. Volkovich and A. Frisch isolated from patients suffering from this disease a microorganism they termed Klebsiella rhinoscleromatis. In 1932, S. Belinov proposed to name the disease Scleroma respiratorium because the pathological process developing in rhinosclerosis may involve not only upper but also lower airways. In 1961, T. Steffen and J. Smith demonstrated that Klebsiella rhinoscleromatis conformed to Koch's postulates and was an etiological factor of inflammatory changes typical of scleroma. This paper presents results of ultrastructural examinations of Klebsiella rhinoscleromatis isolated from two patients with Scleroma respiratorium. It was shown that as the degenerative process developed, the amount of electron-dense material around the bacterial cell wall decreased progressively and only fragments of the cellular membrane remained.

Investigations on the opsonin-dependent interaction of Klebsiella bacteria and human polymorphonuclear leukocytes.

Encapsulated and nonencapsulated Klebsiella strains with different types of fimbriation were tested for their opsonin-dependent stimulation of human polymorphonuclear leukocytes (PMNs). Chemiluminescence (CL) of the PMNs was determined since CL-response can be used indirectly to quantitate phagocytosis. Apparently, the interaction between Klebsiella bacteria and human PMNs is dependent on the type of fimbration and on the presence of capsules. Evaluation of the bactericidal capacity of human PMNs against Klebsiella bacteria positively correlated with the chemiluminescence response demonstrating the importance of fimbriation and capsulation for this process, too.

Transfer and expression of the genetic determinants for O and K antigen synthesis in Escherichia coli O9:K(A)30 and Klebsiella sp. O1:K20, in Escherichia coli K12.

Escherichia coli serotype O9:K(A)30 and Klebsiella O1:K20 produce thermostable capsular polysaccharides or K antigens, which are chemically and serologically indistinguishable. Plasmid pULB113 (RP4::mini-Mu) has been used to mediate chromosomal transfer from E. coli O9:K30 and Klebsiella O1:K20 to a multiply marked, unencapsulated, E. coli K12 recipient. Analysis of the cell surface antigens of the transconjugants confirmed previous reports that the genetic determinants for the E. coli K(A) antigens are located near the his and rfb (O antigen) loci on the E. coli linkage map. The Klebsiella K20 capsule genes were also found to be in close proximity to the his and rfb loci. Electron microscopy revealed significant differences in the structural organization of capsular polysaccharides in these two microorganisms and the morphological differences were also readily apparent in transconjugants expressing the respective K antigens. These results are consistent with the interpretation that at least some of the organizational properties of capsular polysaccharides may be genetically determined, rather than being a function of the outer membrane to which the capsular polysaccharides are ultimately attached.

Bacterial adhesion and pathogenicity.

Bacteria adhere to almost any surface via specific surface molecules of recognition through which a firm union is established for successful colonization of the host. Studies have shown that adhesion plays an important and critical early role in the pathogenesis of infectious diseases, and a series of adhesins have been well documented in a certain number of strains and species of bacteria of medical importance. Attempts have been made to interfere with, or prevent adhesion of, harmful bacteria to the host tissue, using receptor analogues or bacterial adhesin-vaccines as prophylactic measures to protect recipients from specific bacterial diseases. Although much success has been reported from such procedures in laboratory animals and livestock, extensive clinical trials are required to assess the efficacy of such procedures in humans. However, reports from limited studies have shown some encouraging results. Future studies must also be directed to the isolation and characterization of more adhesins and receptors and their specific interactions, which would provide fuller understanding of mechanisms of bacterial adhesion, especially at molecular level.

Opsonin independent interaction of Klebsiella strains with human polymorphonuclear leukocytes.

Nine encapsulated Klebsiella strains with different types of fimbriation and their nonencapsulated mutants were tested for their stimulatory potency for human polymorphonuclear leukocytes in the absence of opsonins. The luminol chemiluminescence assay was used for these experiments. It could be shown that the interaction between Klebsiella bacteria and human leukocytes is rather complex depending not only on the presence of capsules but also on the hydrophobicity of Klebsiella surface and on the type of fimbriation existing.