Bactericidal activity of the organo-tellurium compound AS101 against Enterobacter cloacae.

OBJECTIVES: The antibacterial effect of the organo-tellurium compound AS101 on the Gram-negative bacterium Enterobacter cloacae is shown in this study for the first time. METHODS: The antimicrobial effect of the drug was shown by inhibition of growth, by inhibition of biofilm formation and by its ability to penetrate the bacterial cell and to cause damage and ultrastructural changes. RESULTS: AS101 was found to be a bactericidal drug with MICs and MBCs of 9.4 mg/L. It inhibits bacterial growth and causes a six orders of magnitude decrease in viability in a protein-rich medium, but not in a protein-poorer medium, unless 2-mercaptoethanol is added. Subinhibitory concentrations inhibit motility and biofilm formation. AS101 enters the bacterium through its porins and causes bacterial damage to Na(+)/K(+) pumps and leakage of potassium, phosphorous and sulphur. Ultrastructural changes within the bacterial cell and on its surface demonstrate an incomplete surface with a concavity in the centre that looks like a hole from which aggregates are liberated as well as cell lysis. CONCLUSIONS: AS101 has antibacterial activity, which may be useful against E. cloacae and other species of Enterobacteriaceae as a substitute for current antibiotics that have become ineffective due to increasing bacterial resistance.

Biological characterization of lead-enhanced exopolysaccharide produced by a lead resistant Enterobacter cloacae strain P2B.

A lead resistant bacterial strain isolated from effluent of lead battery manufacturing company of Goa, India has been identified as Enterobacter cloacae strain P2B based on morphological, biochemical characters, FAME profile and 16S rDNA sequence data. This bacterial strain could resist lead nitrate up to 1.6 mM. Significant increase in exopolysaccharide (EPS) production was observed as the production increased from 28 to 108 mg/L dry weight when exposed to 1.6 mM lead nitrate in Tris buffered minimal medium. Fourier-transformed infrared spectroscopy of this EPS revealed presence of several functional groups involved in metal binding viz. carboxyl, hydroxyl and amide groups along with glucuronic acid. Gas chromatography coupled with mass spectrometry analysis of alditol-acetate derivatives of acid hydrolysed EPS produced in presence of 1.6 mM lead nitrate demonstrated presence of several neutral sugars such as rhamnose, arabinose, xylose, mannose, galactose and glucose, which contribute to lead binding hydroxyl groups. Scanning electron microscope coupled with energy dispersive X-ray spectrometric analysis of this lead resistant strain exposed to 1.6 mM lead nitrate interestingly revealed mucous EPS surrounding bacterial cells which sequestered 17 % lead (as weight %) extracellularly and protected the bacterial cells from toxic effects of lead. This lead resistant strain also showed multidrug resistance. Thus these results significantly contribute to better understanding of structure, function and environmental application of lead-enhanced EPSs produced by bacteria. This lead-enhanced biopolymer can play a very important role in bioremediation of several heavy metals including lead.

Carbon : nitrogen : phosphorus ratios influence biofilm formation by Enterobacter cloacae and Citrobacter freundii.

AIMS: To test the effects of C : N : P ratio modification of a well-known nutrient medium formulation, the Endo formulation on biofilm formation by Enterobacter cloacae Ecl and Citrobacter freundii Cf1 in both single-species and binary species biofilms. METHODS AND RESULTS: The C : N : P atom : atom ratio of a well-known nutrient medium formulation, the Endo formulation, that has been applied in fermentative biohydrogen studies, was modified to include two different C concentrations, one containing 17.65 g l(-1) and the other 8.84 g l(-1) sucrose, each containing four different C : N : P ratios, two at higher C : N : P ratios (334 : 84 : 16.8 and 334 : 84 : 3) and two at lower C : N : P ratios (334 : 28 : 5.6 and 334 : 28 : 1). Attached cells were enumerated after dislodging the biofilms that had formed on granular activated carbon (GAC). The modified medium containing 17.65 g l(-1) sucrose and having a C : N : P ratio of 334 : 28 : 5.6 resulted in significantly (P < 0.05) higher counts of attached cells for both single-species biofilms at 7.73 log(10) CFU g(-1) GAC and 9.3 log(10)CFU g(-1) GAC for Ent. cloacae Ecl and Cit. freundii Cf1, respectively, and binary species biofilms at 8.2 log(10) CFU g(-1) GAC and 6.34 log(10) CFU g(-1) GAC for Ent. cloacae Ecl and Cit. freundii Cf1, respectively. Scanning electron micrographs showed qualitative evidence that the 334 : 28 : 5.6 ratio encouraged more complex and extensive biofilm growth for both single-species and binary species biofilms. CONCLUSIONS: The differences in the attachment numbers between the different ratios were found not to be a result of the individual actions of the bacterial isolates involved but rather because of the effects of the various C : N : P ratios. The 334 : 28 : 5.6 ratio showed significantly (P < 0.05) higher counts of attached cells for both single-species and binary species biofilms. SIGNIFICANCE AND IMPACT OF THE STUDY: This study indicates that C : N : P ratios should be a key consideration with regard to maximizing biofilm formation in shake flask and fluidized bed bioreactor studies as well as understanding fundamental factors affecting biofilm growth in natural environments.

Rapid contrasting of ultrathin sections using microwave irradiation with heat dissipation.

The use of microwave irradiation (MWI) to accelerate fixation, dehydration and contrasting (staining) for electron microscopy has been applied to the development of rapid methods to process biological samples in electron microscopy. A simple explanation is that the reduced time in those procedures is due to heating. In this paper we propose a contrasting method for thin sections that avoids the thermal effects of MWI. Grids with thin sections of mouse kidney, the dinoflagellate Alexandrium monilatum, spermatophores of the fly Archicepsis diversiformis, the bacteria Acinetobacter calcoaceticum and Enterobacter cloacae were placed into Beem capsules and stained with uranyl acetate and lead citrate, while immersed in an ice-water bath, and irradiated for periods ranging from 30 s to 2 min. After each contrasting procedure, the Beem capsule was filled with distilled water to wash the grids under MWI with the same irradiation time as used to contrast. Good results were obtained on irradiating for 1 min and the temperature of the Beem capsule was maintained around 5 degrees C.

Differential distributions in tissues and efficacies of aztreonam and ceftazidime and in vivo bacterial morphological changes following treatment.

The differential tissue distributions of aztreonam and ceftazidime within fibrin clots infected with Pseudomonas aeruginosa, Enterobacter cloacae, and Serratia marcescens, their efficacies, and the in vivo bacterial morphological changes induced by these drugs were evaluated. Rabbits were given intravenously a single dose of 100 mg of either agents/kg of body weight. In the cores of the clots, the peak levels of both drugs were much lower than those observed in the peripheries and in serum. Aztreonam's half-lives within the peripheries and in the cores of the fibrin clots were up to six times higher than observed in serum, while ceftazidime's half-lives in clots were twice that observed in serum. This resulted in a much greater penetration ratio for aztreonam than for ceftazidime. Both drugs controlled the growth of P. aeruginosa in vivo, but E. cloacae and S. marcescens responded better to ceftazidime. Morphological changes were more abundant in the peripheries than in the cores of the clots. In the control group, P. aeruginosa's morphology in the cores was different than that in the peripheries of the clots. Against P. aeruginosa, aztreonam did induce morphological changes in the cores while ceftazidime did not. Electron microscopic studies revealed that morphological changes associated with aztreonam seemed different than those of ceftazidime. Along with elongation of bacteria, more bow tie and herniated bacteria were observed with aztreonam. Though both agents selectively affect PBP 3, as manifested by elongated bacteria, they induce in the peripheries of the clots thickening, breaks, and detachment in bacterial cell walls, alterations which are generally associated with antibiotics affecting PBP 1a and 1b.

Bacteriocins: nature, function and structure.

Bacteriocins are extracellular substances produced by different types of bacteria, including both Gram positive and Gram negative species. They can be produced spontaneously or induced by certain chemicals such as mitomycin C. They are biologically one of the important substances, and have been found to be useful in membrane studies and also in typing pathogenic microorganisms causing serious nosocomial infections. Bacteriocins are a heterogeneous group of particles with different morphological and biochemical entities. They range from a simple protein to a high molecular weight complex: the active moiety of each molecule in all cases seems to be protein in nature. The genetic determinants of most of the bacteriocins are located on the plasmids, apart from few which are chromosomally encoded. These bactericidal particles are species specific. They exert their lethal activity through adsorption to specific receptors located on the external surface of sensitive bacteria, followed by metabolic, biological and morphological changes resulting in the killing of such bacteria. This review summarises the classification, biochemical nature, morphology and mode of action of bacteriocins as well as their genetic determinants and the microbiological relevance of these bactericidal agents.

[Plasmid size determination using 3 methods]. / Determinação do tamanho de plasmídios por três métodos.

Analysis of bacterial plasmid profiles has been shown to be very important in epidemiological studies, especially those involving outbreaks of nosocomial infections. The molecular weight size of unknown plasmids is determined by comparing their band pattern obtained in agarose gel electrophoresis with those obtained with plasmids that have been used as molecular weight or size standards. In this study, we determined the size of plasmids present in clinical samples of Enterobacter cloacae comparing their electrophoresis mobility with seven plasmids of known size, using three different mathematical methods. For plasmids with molecular weight ranging from 2 kb to 100 kb. The most accurate determinations were obtained by power-function. Analyses using the exponential variables obtained with these plasmids were accurate for two types of plasmids, those with size ranging from 50 kb to 100 kb and those with size ranging from 2 kb to 30 kb. We also observed discrepancies among the methodologies described, including one used by a computer software designed for calculating the size of plasmids DNA.

Purification and characterization of extracellular alginate lyase from Enterobacter cloacae M-1.

An alginate lyase from the culture supernatant of Enterobacter cloacae M-1 was purified by ammonium sulfate precipitation, cation-exchange chromatography (SP-Toyopearl), and gel filtration (Ultrogel AcA44). The final preparation thus obtained showed a single band on SDS-PAGE. The purified enzyme had the molecular weight of 38,000 and 32,000 by SDS-PAGE and gel filtration, respectively. The pI of the enzyme was 8.9. The optimum pH and temperature for the enzyme reaction were around 7.8 and 30 degrees C, respectively. The enzyme was unstable on heating. EDTA completely inhibited the enzyme activity, but the activity was completely restored by the treatment with CaCl2. The enzyme was specific for poly-guluronate and produced several kinds of unsaturated oligomers from the gluluronate. This suggested that the enzyme could be classified as an endo poly-guluronate lyase.

Antibiotic-induced release of endotoxin from bacteria in vitro.

The ability of cefotaxime, ciprofloxacin, piperacillin and tobramycin to cause release of endotoxin was examined in vitro with cultures of Enterobacter cloacae and Escherichia coli. Endotoxin was measured by a quantitative limulus amoebocyte lysate assay and its presence was confirmed by silver staining of the lipopolysaccharide moiety following SDS-PAGE. The morphology of the bacteria during antibiotic exposure was examined by scanning electronmicroscopy. Cefotaxime, ciprofloxacin and piperacillin caused significant endotoxin release, correlating with their ability to affect cell-wall morphology, causing filamentation, wall breakage and cell lysis. In contrast, little endotoxin was released when bacteria were exposed to tobramycin and no morphological changes were observed when bacteria were exposed to bactericidal concentrations of this aminoglycoside. Its antimicrobial spectrum and bactericidal activity make tobramycin an appropriate agent for treatment of sepsis caused by gram-negative bacteria and its lack of propensity to elicit excessive release of endotoxin may avoid exacerbation of endotoxin-related shock in sepsis.

Investigation of cell envelope damage to Pseudomonas aeruginosa and Enterobacter cloacae by dibromopropamidine isethionate.

Electron micrographs of log-phase Pseudomonas aeruginosa and Enterobacter cloacae cultured for 4 h in the presence of subinhibitory concentrations of dibromopropamidine isethionate indicate that this antibacterial agent can cause marked damage to the cell envelope structures of both species. This result provides an explanation of how dibromopropamidine can enhance the uptake and thus the activity of a second antibacterial agent used in combination with it.

Peptidoglycan tripeptide content and cross-linking are altered in Enterobacter cloacae induced to produce AmpC beta-lactamase by glycine and D-amino acids.

Induction of AmpC beta-lactamase in Enterobacter cloacae ATCC 13047 by D-methionine, glycine, or D-tryptophan was accompanied by alterations in peptidoglycan composition and structure; in the case of D-methionine, it was also accompanied by morphologic changes. A decrease in peptidoglycan tripeptides was seen. With glycine, there was an increase in the proportion of diaminopimelic-diaminopimelic cross-links. The possible implications of these changes for beta-lactamase induction are discussed.

Phospholipid impregnation of abdominal rubber drains: resistance to bacterial adherence but no effect on drain-induced bacterial translocation.

In order to evaluate the effect of surface modification of biomaterials on bacterial adherence and bacterial translocation after intraperitoneal biomaterial implantation, phosphatidylcholine- or phosphatidylinositol-impregnated rubber drain pieces, which had been intraperitoneally implanted in the rat for 2 and 7 days, or unimplanted, were incubated in vitro with 3H-labelled Escherichia coli and Enterobacter cloacae. As compared with unimpregnated pieces, the adherence of bacteria significantly decreased to phosphatidylcholine- and phosphatidylinositol-impregnated rubber drain pieces that were either unimplanted or implanted for 2 days, but not for 7 days. The supplementation of albumin in the medium reduced the adherence of bacteria to the unimplanted, unimpregnated drain pieces, but did not further decrease adherence of bacteria to the unimplanted, phospholipid-impregnated brain pieces. Bacterial growth was inhibited after incubation in nutrient broth supplemented with phospholipids. The incidence of enteric bacterial translocation induced by intraperitoneal drain implantation did not differ between phospholipid-impregnated and unimpregnated drain pieces. Scanning electron microscopy revealed a large amount of biofilm and fibrous deposition on the surface of the implanted, phospholipid-impregnated rubber drain pieces. Thus, phospholipid impregnation of rubber drains reduces bacterial adherence and inhibits bacterial growth, without influencing the incidence of bacterial translocation.

An electronmicroscope study of the effect of sulphadiazine and trimethoprim on Enterobacter cloacae.

Electronmicroscopy of thin sections of log phase cells of Enterobacter cloacae NCTC 10005 grown for 4 h in the presence of sulphadiazine 250 micrograms/ml, trimethoprim 12.5 microliters/ml or the combination of sulphadiazine 250 micrograms/ml plus trimethoprim 12.5 micrograms/ml indicated that both agents caused marked morphological damage even though the MIC of sulphadiazine for the E. cloacae strain was > 3000 micrograms/ml. The damage took the form of electron-transparent areas devoid of ribosomes in the cytoplasm and detachment of the outer membrane. The latter was most marked with trimethoprim, which also caused damage to the cytoplasmic membrane. It is postulated that the synthesis of the peptidoglycan layer was affected by the antimetabolites since the morphological effects were strikingly similar to those caused by treatment of E. cloacae with disodium edetate plus lysozyme. Viable counts of cultures undergoing the same treatments as those prepared for electronmicroscopy indicated that although sulphadiazine merely partially inhibited growth it nevertheless enhanced the bactericidal action of trimethoprim over a 5-h period.

Diffusion of beta-lactam antibiotics into proteoliposomes reconstituted with outer membranes of isogenic imipenem-susceptible and -resistant strains of Enterobacter cloacae.

The influence of outer membrane (OM) permeability on carbapenem susceptibility was studied in strains of Enterobacter cloacae, a species in which carbapenem resistance depends upon the conjunction of overproduction of the chromosomal cephalosporinase and reduction of OM permeability. Relative trans-OM diffusion rates were measured using the liposome swelling assay. Proteoliposomes were reconstituted with OM from the members of an isogenic set of E. cloacae strains, selected in vivo or in vitro, which produced either porins F and D (wild-type), or F or D only, or neither. For all but one mutant, and compared with the wild-type strain, the respective increases in MICs and decreases in trans-OM diffusion of carbapenems were: nil and 13 to 18%; 4- to 32-fold and 33 to 50%; > or = 64-fold and > or = 90%. Our results suggest (i) that carbapenems (and other beta-lactam antibiotics) diffuse through porins F and D, but more rapidly through porin F, and (ii) that OM permeability is the critical factor in determining the level of MICs of carbapenems for cephalosporinase-overproducing strains of E. cloacae. The OM of one particular low-level carbapenem-resistant and porin F- and D-deficient mutant was at least five times more permeable to carbapenems than the similarly porin-deficient high-level resistant mutants. We infer from this observation the possible existence of an alternative carbapenem penetration pathway which could be associated with two as yet uncharacterized overproduced OM proteins of about 22 and 47 kDa.

Localization of cephalosporinase in Enterobacter cloacae by immunocytochemical examination.

Enterobacter cloacae NUH10 was isolated at Nagasaki University Hospital in 1987. E. cloacae NUH10 is a mutant strain which produces high levels of cephalosporinase. E. cloacae ATCC 23355 is known to be sensitive to so-called third generation cephems and produces an inducible cephalosporinase. The polyclonal antibody to cephalosporinase extracted from E. cloacae NUH10 was utilized in post-embedding immunogold labeling in order to localize this protein in E. cloacae ATCC 23355 and E. cloacae NUH10. Immunocytochemical localization of the cephalosporinase in both strains was observed with and without incubation with an inducer. Cephalosporinase was detected in both the cytoplasm and periplasmic space of E. cloacae ATCC 23355 and E. cloacae NUH10 incubated in medium including cefoxitin as an inducer. In the case of incubation without the inducer, a small quantity of cephalosporinase was located in the periplasmic space in either strain of bacteria. Western blot analysis showed that cephalosporinase was predominantly localized in the periplasmic space rather than in the cytoplasmic space.