Antibacterial properties of Pseudomonas aeruginosa immunotype 1 lipopolysaccharide-specific monoclonal antibody (MAb) in a murine thigh infection model: combined effects of MAb and ceftazidime

A murine monoclonal antibody (MAb) specific for the Pseudomonas aeruginosa immunotype 1 (It-1) lipopolysaccharide (LPS) O-side chain was evaluated in terms of its in vitro bactericidal opsonophagocytic activity and in vivo bacterial killing in a mouse thigh infection model. An immunoglobulin (Ig) G2a MAb Ld3-2F2, specific for It-1 LPS, mediated in vitro complement-dependent opsonophagocytic killing at a concentration of 10 microg/ml. MAb-mediated, complement-dependent killing also occurred in the absence of neutrophils at serum concentrations in excess of 20%. A remarkable synergy was observed in opsonophagocytic assays between MAb Ld3-2F2 (0.5 microg/ml) and ceftazidime (1/4 MIC). The administration of MAb Ld3-2F2 at a level of 1 microg resulted in a significant decrease in the number of bacteria in the thigh muscles of normal mice, while 100 microg of the same MAb was required for one log of reduction in the number of bacteria at the same site in neutropenic mice. The combined therapy with MAb Ld3-2F2 and ceftazidime provided a significant reduction in the density of bacteria in the thigh muscle at 9 hr post-infection in normal and neutropenic mice as compared with those after treatment alone or with no treatment (P< 0.01). These favorable in vitro and in vivo interactions of an LPS-specific IgG MAb and ceftazidime strongly support their potential for use in therapy, combined with an LPS-reactive MAb and parenteral antipseudomonas beta-lactam antibiotics in the therapy of systemic Pseudomonas infections in normal and neutropenic hosts.     

Effect of sulbactam on anti-pseudomonal activity of beta-lactam antibiotics in cells producing various levels of the MexAB-OprM efflux pump and beta-lactamase

The beta-lactamase inhibitor, sulbactam, was tested for beta-lactamase inhibitory activity in Pseudomonas aeruginosa cells producing various levels of both the MexAB-OprM efflux pump and beta-lactamase. We found that sulbactam lowered the MICs of cefoperazone and piperacillin by inhibiting the beta-lactamase 8-fold in the cell producing a constitutively high level of AmpC-type beta-lactamase and a wild-type level of MexAB-OprM pump compared with that without sulbactam. The MICs of cefoperazone and piperacillin in the cell producing a constitutively high level of both the efflux pump and beta-lactamase under the presence of sulbactam were 8 and 4 times, respectively, lower than that without sulbactam. The MICs of sulbactam in the cell producing a constitutively high and a wild-type level of the efflux pump were 16- and 8-fold higher, respectively, than that in the mutant lacking the efflux pump. We concluded that sulbactam exerts potent beta-lactamase inhibitory activity in the cell producing a high level of efflux pump, in spite of the fact that sulbactam serves as a substrate of the MexAB-OprM pump. Increasing amounts of sulbactam over the weight of beta-lactams further strengthen the effect of beta-lactam antibiotics.     

Evaluation of the biofilm-forming ability and genetic typing for clinical isolates of Pseudomonas aeruginosa by enterobacterial repetitive intergenic consensus-based PCR

Biofilm formation is an important phenotype associated with chronic Pseudomonas aeruginosa infections. In the present study, a total of 48 P. aeruginosa strains isolated from clinical specimens were examined for their biofilm-forming ability using a microtiter plate method. The different biofilm-forming abilities were demonstrated among the strains; however, most strains formed a larger biofilm than strain PAO1, a reference strain. The genetic typing was also carried out by enterobacterial repetitive intergenic consensus-based polymerase chain reaction. Although they were divided into five groups (A to E), most of the strains showing the higher biofilm-forming ability were found to be in groups D and E, suggesting a significant relationship between the biofilm-forming ability and the genetic group.     

The new approaches to whole genome analysis of bacteria

A range of recombinant DNA techniques now enables whole genome analysis of any bacterium to be carried out without recourse to the classical means of bacterial genetic exchange. Using enzymes which cut infrequently, such as SpeI, combined with pulsed field gel electrophoresis, a physical map of ordered fragments can be constructed. By means of cloned fragments of known genes or oligonucleotides synthesized using data from DNA or protein sequence banks, the location of individual genes on this map can be determined. We have used these techniques to study whole genome structure in three species of Pseudomonas: P. aeruginosa, P. putida and P. solanacearum.     

The Potential of the Ni-Resistant TCE-Degrading Pseudomonas putida W619-TCE to Reduce Phytotoxicity and Improve Phytoremediation Efficiency of Poplar Cuttings on A Ni-TCE Co-Contamination

To examine the potential of Pseudomonas putida W619-TCE to improve phytoremediation of Ni-TCE co-contamination, the effects of inoculation of a Ni-resistant, TCE-degrading root endophyte on Ni-TCE phytotoxicity, Ni uptake and trichloroethylene (TCE) degradation of Ni-TCE-exposed poplar cuttings are evaluated.

After inoculation with P. putida W619-TCE, root weight of non-exposed poplar cuttings significantly increased. Further, inoculation induced a mitigation of the Ni-TCE phytotoxicity, which was illustrated by a diminished exposure-induced increase in activity of antioxidative enzymes. Considering phytoremediation efficiency, inoculation with P. putida W619-TCE resulted in a 45% increased Ni uptake in roots as well as a slightly significant reduction in TCE concentration in leaves and TCE evapotranspiration to the atmosphere.

These results indicate that endophytes equipped with the appropriate characteristics can assist their host plant to deal with co-contamination of toxic metals and organic contaminants during phytoremediation. Furthermore, as poplar is an excellent plant for biomass production as well as for phytoremediation, the obtained results can be exploited to produce biomass for energy and industrial feedstock applications in a highly productive manner on contaminated land that is not suited for normal agriculture. Exploiting this land for biomass production could contribute to diminish the conflict between food and bioenergy production.     

EFFECT OF METAL TOLERANT PLANT GROWTH PROMOTING BACTERIA ON GROWTH AND METAL ACCUMULATION IN ZEA MAYS PLANTS GROWN IN FLY ASH AMENDED SOIL

The present study was undertaken to examine the effect of the application of fly ash (FA) into Garden soil (GS), with and without inoculation of plant growth promoting bacteria (PGPB), on the growth and metal uptake by Zea mays plants. Three FA tolerant PGPB strains, Pseudomonas sp. PS5, PS14, and Bacillus sp. BC29 were isolated from FA contaminated soils and assessed for their plant growth promoting features on the Z. mays plants. All three strains were also examined for their ability to solubilize phosphate and to produce Indole Acetic Acid (IAA), siderophores, and hydrogencynide acid (HCN) production. Although inoculation of all strains significantly enhanced the growth of plants at both the concentration of FA but maximum growth was observed in plants inoculated with BC29 and PS14 at low level (25%) of FA concentration. The experimental results explored the plant growth promoting features of selected strains which not only enhanced growth and biomass of plants but also protected them from toxicity of FA.     

计算机辅助设计联合斑点杂交筛选铜绿假单胞菌PAO1 motA基因反义寡核苷酸序列

为了筛选出能与铜绿假单胞菌PAO1 motA基因的mRNA结合紧密的反义寡核苷酸序列,采用全基因寻靶技术（full length gene targeting,FLGT）,运用计算机软件（Mfold和RNA Structure4.6）模拟铜绿假单胞菌PAO1 motA基因mRNA的二级结构,根据最小自由能原理设计出8条寡核苷酸探针序列;PCR扩增出全长motA基因,克隆motA基因并进行体外转录,同时用地高辛标记mRNA,以斑点杂交方法筛选出与motA基因mRNA结合紧密、杂交信号较强的寡核苷酸序列。斑点杂交结果显示8条寡核苷酸中的4条有较强的杂交信号,从而成功筛选到了能与motA mRNA牢固结合的反义序列,为进一步研究以motA基因为靶的反义技术抑制生物膜形成打下基础。     

Lithocholic Acid Side-chain Cleavage to Produce 17-Keto or 22-Aldehyde Steroids by Pseudomonas putida strain ST-491 Grown in the Presence of an Organic Solvent,Diphenyl Ether

We devised a method to screen for microorganisms capable of growing on bile acids in the presence of organic solvents and producing organic solvent-soluble derivatives. Pseudomonas putida biovar A strain ST-491 isolated in this study produced decarboxylated derivatives from the bile acids. Strain ST-491 grown on 0.5% lithocholic acid catabolized approximately 30% of the substrate as a carbon source, and transiently accumulated in the medium androsta-1,4-diene-3,17-dione in an amount of corresponding to 5% of the substrate added. When 20% (v/v) diphenyl ether was added to the medium, 60% of the substrate was converted to 17-keto steroids (androst-4-ene-3,17-dione-like steroid, androsta-1,4-diene-3,17-dione) or a 22-aldehyde steroid (pregna-1,4-dien-3-on-20-al). Amounts of the products were responsible for 45, 10, and 5% of the substrate, respectively. In the presence of the surfactant Triton X-100 instead of diphenyl ether, 40% of the substrate was converted exclusively to androsta-1,4-diene-3,17-dione.     

Functions of Malonate Decarboxylase Subunits from Pseudomonas putida

Malonate decarboxylase from Pseudomonas putida is composed of five subunits, α, β, γ, δ, and ε. Two subunits, δ and ε, have been identified as an acyl-carrier protein (ACP) and malonyl-CoA:ACP transacylase, respectively. Functions of the other three subunits have not been identified, because recombinant subunits expressed in Escherichia coli formed inclusion bodies. To resolve this problem, we used a coexpression system with GroEL/ES from E. coli, and obtained active recombinant subunits. Enzymatic analysis of the purified recombinant subunits showed that the α subunit was an acetyl-S-ACP:malonate ACP transferase and that the βγ-subunit complex was a malonyl-S-ACP decarboxylase.