Motility effects biofilm formation in Pseudomonas aeruginosa and Enterobacter cloacae.

Chronic infections caused by gram negative bacteria are the mains reasons to have morbidity and death in patients, despite using high doses of antibiotics applied to cure diseases producing by them. This study was designed to identify the role of flagella in biofilm formation Ten pure strains were collected from our lab. Morphological variation and motility assays led us to study two strains in detail. They were characterized biochemically, physiologically and genetically. Biofilm formation analysis was performed using test tube assay, congo red assay and liquid-interface coverslip assay. In order to disrupt flagella of studied strains, blending was induced for 5, 10 and 15 minutes followed by centrifugation and observing motility using motility test. Biofilm quantification of wild type (parental) and blended strains was done using test tube and liquid interface coverslip assays. 16S rRNA sequencing identified strains as Pseudomonas aeroginosa and Enterobacter cloacae. Significant biofilm formation (p>0.05) by was observed after 72 and 18 hours using test tube and liquid-interface coverslip assays respectively. Flagellar disruption showed that 15 minutes blending caused significant reduction in both strains, hence demonstrated that flagellar mediated motility could be a potent strategy to stabilize aggregate and invest resources for biofilm formation in P. aeruginosa and E. cloacae.

Bioadsorption and biostabilization of cadmium by Enterobacter cloacae TU.

Biostabilization of cadmium, a hazardous chemical found widely in China, was attempted using Enterobacter cloacae TU (E.cloacae TU). A cadmium (Cd)-tolerant E.cloacae TU was obtained by mutagenesis using an atmosphere pressure glow discharge plasma system, and it displayed regular growth behavior in the presence of 250 mg/L Cd in solution. The maximum stabilization capacity of E.cloacae TU toward Cd reached 67.0 ± 3.5 mg/g dry cell weight at an initial Cd concentration of 200 mg/L. The percentage of Cd removal by E.cloacae TU reached 97.4± 0.3% at an initial Cd concentration of 20 mg/L. A desorption experiment confirmed both extracellular adsorption and intracellular uptake contribute to biostabilization, although Cd was mainly distributed on the surface of E.cloacae TU cells due to over-secretion of extracellular polysaccharides under Cd stimulus. The changes in morphology and functional groups of the E.cloacae TU cell surface in the presence of Cd were analyzed using X-ray Photoelectron Spectroscopy (XPS), Scanning Electron Microscope (SEM), Transmission Electron Microscope (TEM) and Fourier Transform Infrared Spectoscopy (FT-IR). The feasibility of using E.cloacae TU for this purpose was further confirmed by on site remediation, in which the application of E.cloacae TU reduced the bioavailability and moreover the accumulation of Cd in tobacco plants without affecting the quality of flue-cured tobacco.

Antibacterial active compounds from Hypericum ascyron L. induce bacterial cell death through apoptosis pathway.

Hypericum ascyron L. has been used as a traditional medicine for the treatment of wounds, swelling, headache, nausea and abscesses in China for thousands of years. However, modern pharmacological studies are still necessary to provide a scientific basis to substantiate their traditional use. In this study, the mechanism underlying the antimicrobial effect of the antibacterial activity compounds from H. ascyron L. was investigated. Bioguided fractionation of the extract from H. ascyron L. afforded antibacterial activity fraction 8. The results of cup plate analysis and MTT assay showed that the MIC and MBC of fraction 8 is 5 mg/mL. Furthermore, using Annexin V-FITC/PI, TUNEL labeling and DNA gel electrophoresis, we found that cell death with apoptosis features similar to those in eucaryon could be induced in bacteria strains after exposure to the antibacterial activity compounds from H. ascyron L. at moderate concentration. In addition, we further found fraction 8 could disrupt the cell membrane potential indicate that fraction 8 exerts pro-apoptotic effects through a membrane-mediated apoptosis pathway. Finally, quercetin and kaempferol 3-O-ß-(2″-acetyl)-galactopyranoside, were identified from fraction 8 by means of Mass spectrometry and Nuclear magnetic resonance. To our best knowledge, this study is the first to show that Kaempferol 3-O-ß-(2″-acetyl)-galactopyranoside coupled with quercetin had significant antibacterial activity via apoptosis pathway, and it is also the first report that Kaempferol 3-O-ß-(2″-acetyl)-galactopyranoside was found in clusiacea. Our data might provide a rational base for the use of H. ascyron L. in clinical, and throw light on the development of novel antibacterial drugs.

Reduction of vanadium(V) by Enterobacter cloacae EV-SA01 isolated from a South African deep gold mine.

Dissimilatory reduction of vanadium(V) by Enterobacter cloacae EV-SA01, isolated from a gold mine at 1.6 km below surface, is shown to occur anaerobically as well as aerobically. Growth rates were unaffected by up to 2 mM V(2)O(5). Reduction of vanadium(V) was growth phase-dependent and resulted in cell deformities and precipitation of the vanadium in its lower oxidation states. The vanadate reductase activity was membrane-associated and coupled the oxidation of NADH to the reduction of vanadate.

Growth of cell-wall-deficient variants of Enterobacter cloacae facilitates beta-lactamase derepressed mutants.

The degree to which cell-wall-deficient bacteria (CWDB) are involved in the generation of beta-lactamase derepressed mutants (DM) was measured using Enterobacter cloacae 3624. The frequency of DM in non-permissive isotonic ticarcillin medium was compared with their frequency in hypertonic ticarcillin medium that supports CWDB growth. DM were resistant to extended spectrum penicillins and cephalosporins and had a basal beta-lactamase activity of >300 units/mg protein. Anaerobic growth of CWDB increased the relative risk of DM 2 x 10(6)-fold. Aerobic incubation produced fewer CWDB colonies but the risk of DM was still increased 400-fold over non-permissive controls. These results define a new role for CWDB as intermediaries in the emergence of resistance.

Further studies on RpoS in enterobacteria: identification of rpoS in Enterobacter cloacae and Kluyvera cryocrescens.

RpoS, the alternative sigma factor sigma(s), is important for bacterial survival under extreme conditions. Many enterobacteria are opportunistic human pathogens and their ability to survive in a changing environment could be an essential step for their virulence. To determine the presence of this gene in enteric bacteria, an Escherichia coli rpoS probe was constructed and used to detect the presence of this gene in different species. A gene homologous to rpoS was found in Citrobacter amalonaticus, Enterobacter cloacae, Klebsiella planticola, Kluyvera cryocrescens, Serratia rubidaea, Shigella sonnei, and Yersinia ruckeri. Providencia stuartii and Proteus vulgaris were the only tested enterobacteria that did not show any signal with the E. coli rpoS probe or that did not lead to amplification of an rpoS fragment using specific primers. The rpoS gene from E. cloacae and from K. cryocrescens was cloned and sequenced and a mutant allele was constructed in E. cloacae. Survival rates under different harsh conditions were followed in order to determine the effect of rpoS inactivation in exponential- and stationary-phase cells of both strains. E. cloacae rpoS mutants were more sensitive to extreme pH, high osmolarity, and high temperature than the wild-type.

Enterobacter cloacae is an endophytic symbiont of corn.

The bacterium Enterobacter cloacae is presently used for biocontrol of postharvest diseases of fruits and vegetables and as a preplant seed treatment for suppression of damping-off. This bacterium has apparent affinities for several grass species, but it is not considered to be an endophyte. While screening corn for fungi and bacteria with potential for biocontrol of various corn diseases, the surface-sterilized kernels of one unknown Italian corn cultivar produced fungus-free corn seedlings with roots endophytically infected by E. cloacae. This paper describes the microscopic nature of E. cloacae RRC 101 with corn, and the in vitro control of Fusarium moniliforme and other fungi with this bacterium. Light and electron microscopy determined that this isolate of E. cloacae was biologically associated with corn seedling roots, where it was distributed intercellularly within the cortex and stele. This is a first report of a strain of this bacterium as an endophytic symbiont of roots. Following a topical application of E. cloacae to kernels, and upon germination this bacterium readily infected roots of two other corn cultivars. The bacterium was observed within the endosperm of germinating corn seedling, but germination was not affected. Further, the bacterium was isolated from leaves and stems of 3- to 6-week-old seedlings indicating that the above ground portions of corn were also colonized. There was no evidence of damage to cells of the root during a three to four week observation period. This bacterium was antagonistic to several isolates of the corn pathogen Fusarium moniliforme, and to two other species of fungi, all of which produce mycotoxins on corn.

Separation and quantification of murein and precursors from Enterobacter cloacae after treatment with trimethoprim and sulphadiazine.

The intracellular concentrations of the soluble murein precursors UDP-Mur-NAc-pentapeptide in the cytoplasm, the membrane-bound lipid precursor disaccharide pentapeptide and the muropeptides of Enterobacter cloacae cultures treated with trimethoprim (12.5 micrograms mL-1) and sulphadiazine (250 micrograms mL-1) were determined by using capillary zone electrophoresis analysis. In the presence of trimethoprim, UDP-Mur-NAc-pentapeptide as well as disaccharide pentapeptide accumulated. In the case of sulphadiazine-treated cells, the concentration of UDP-Mur-NAc-pentapeptide roughly paralleled the control cells but sulphadiazine caused a slow incremental accumulation of disaccharide pentapeptide. The muropeptide composition of the murein indicated that the differences between the peptidoglycans produced by the control cells and the cells grown in the presence of either trimethoprim or sulphadiazine alone or in combination were quite marked. The results suggest that the enhanced activity of trimethoprim plus sulphadiazine against E. cloacae is caused by an additional effect on the inhibition of the bacterial peptidoglycan biosynthesis and that this additional effect is a fundamental part of the antibacterial action of the antimetabolites. This effect leads to changes of cell morphology and resultant changes in bacterial cell permeability.

Effects on cell morphology of growing Pseudomonas aeruginosa, Enterobacter cloacae and Staphylococcus aureus in subinhibitory concentrations of p-aminobenzoic acid.

Light microscopy and electron microscopy studies with Enterobacter cloacae, Pseudomonas aeruginosa and Staphylococcus aureus grown in the presence of subinhibitory concentrations of p-aminobenzoic acid indicate that p-aminobenzoic acid may have a direct or indirect effect on the cell walls. The morphological effects of p-aminobenzoic acid varied with different bacterial species. E. cloacae grown in the presence of p-aminobenzoic acid produced filaments while P. aeruginosa cells were elongated and had thicker peripheral cell walls. S. aureus had increased overall cell size and thicker transverse cell walls.