Incidence of antibiotic-resistant Klebsiella pneumoniae and Enterobacter species in freshwater wetlands

AIMS: The aim of this study was to assess the incidence of Enterobacteriaceae (potential human and animal pathogens) in wetlands. METHODS: Enterobacteriaceae, selected from the sediments and rhizosphere of wetland plant Juncus effusus L., were analysed using classical microbiological methods, API20E, API20NE, fatty acid analyses, and 16S rRNA sequencing. Assessed virulence factors include antibiotic resistance, presence of plasmids and capsules. RESULTS: Klebsiella pneumoniae, Enterobacter cloacae and Enterobacter asburiae, known human pathogens, were identified. K. pneumoniae 16S rRNA gene sequence showed the significant hit (E < 0.001) with the unculturable bacteria obtained from faeces of elderly individuals (accession number AB099804) when Genbank database was used. Ent. asburiae 16S rRNA gene sequence showed the significant hit with (E < 0.001) with the unculturable bacteria obtained from the pig gastrointestinal tract (accession number AF371852). The rate of antibiotic resistance (<50 microg ml(-1)) was high for ampicillin and cephalosporins for the most strains (75.7%) yet low (>10 to 20 microg ml(-1)) for kanamycin, tetracycline and chloramphenicol for all strains tested. Capsules were detected in all investigated strains. PCR detected membrane protein but not chromosomally encoded beta-lactamase. Significance and Impact of the Study: The antibiotic resistance of tested strains and presence of capsules (protect micro-organisms from phagocytosis) suggest that wetland sediments and rhizosphere present a potential reservoirs for enteric human and animal pathogens.     

Molecular cloning, characterization, and overexpression of a novel [Fe]-hydrogenase isolated from a high rate of hydrogen producing Enterobacter cloacae IIT-BT 08

Degenerate primers were designed from the conserved zone of hydA structural gene encoding for catalytic subunit of [Fe]-hydrogenase of different hydrogen producing bacteria. A 750 bp of PCR product was amplified by using the above-mentioned degenerate primers and genomic DNA of Enterobacter cloacae IIT-BT 08 as template. The amplified PCR product was cloned and sequenced. The sequence showed the presence of an ORF of 450 bp with significant similarity (40%) with C-terminal end of the conserved zone (H-cluster) of [Fe]- hydrogenase. hydA ORF was then amplified and cloned in-frame with GST in pGEX4T-1 and overexpressed in a non-hydrogen producing Escherichia coli BL-21 to produce a GST-fusion protein of a calculated molecular mass of about 42.1 kDa. Recombinant protein was purified and specifically recognized by anti-GST monoclonal antibody through Western blot. Southern hybridization confirmed the presence of this gene in E. cloacae IIT-BT 08 genome. In vitro hydrogenase assay with the overexpressed hydrogenase enzyme showed that it is catalytically active upon anaerobic adaptation. In vivo hydrogenase assay confirmed the presence of H2 gas in the gas mixture obtained from the batch culture of recombinant E. coli BL-21. A tentative molecular mechanism has been proposed about the transfer of electron from electron donor to H-cluster without the mediation of the F-cluster.     

Stable integration and expression of mosquito-larvicidal genes from Bacillus thuringiensis subsp. israelensis and Bacillus sphaericus into the chromosome of Enterobacter amnigenus: a potential breakthrough in mosquito biocontrol

Previously, we have successfully integrated a spectinomycin/streptomycin resistance gene into Enterobacter amnigenus strain An11, a potential host for mosquito control, using in vivo recombination via homologous recombination (An11S4::Omega). We now report the successful transfer of two mosquito-larvicidal genes, cry4B from Bacillus thuringiensis subsp. israelensis and binary toxin genes from Bacillus sphaericus, into the host genome. To facilitate the screening procedure, the E. amnigenus derivative, An11S4::Omega, was used as a host. The integration of both toxin genes by two successive crossover events interrupted the Omega region yielding two integrants designated An11S4::cry4B and An11S4::Omega::bin, respectively. Differences in the integration efficiency of these toxin genes were observed. The presence of both genes in the target sites of the host genome was verified by PCR. Cry4B was expressed weakly from An11S4::cry4B, but no expression of the binary toxin gene could be detected from An11S4::Omega::bin. Nevertheless, these two integrants exhibited mosquito-larvicidal activity against Aedes and Culex, suggesting that both proteins were expressed, but at very low levels.     

Inhibitors of antibiotic efflux pump in resistant Enterobacter aerogenes strains

Enterobacter aerogenes, a nosocomial pathogen, is frequently exhibiting multidrug resistance mechanisms associated with a change in membrane permeability. In clinical isolates, active efflux plays a prominent role in antibiotic resistance. We report here the effect of three unrelated compounds that are able to restore a noticeable antibiotic susceptibility to resistant strains. The targeting of various parameters which contribute to the efficacy of the efflux mechanism, such as energy, flux selectivity, or functional assembly of the membrane complex, increases the intracellular chloramphenicol concentration in resistant isolates.     

Chloramphenicol and expression of multidrug efflux pump in Enterobacter aerogenes

Chloramphenicol has been reported to act as an inducer of the multidrug resistance in Escherichia coli. A resistant variant able to grow on plates containing 64 microg/ml chloramphenicol was obtained from the Enterobacter aerogenes ATCC 13048-type strain. Chloramphenicol resistance was due to an active efflux of this antibiotic and it was associated with resistance to fluoroquinolones and tetracycline, but not to aminoglycoside or beta-lactam antibiotics. MDR in the chloramphenicol-resistant variant is linked to the overexpression of the major AcrAB-TolC efflux system. This overexpression seems unrelated to the global Mar and the local AcrR regulatory pathways.     

Description of Enterobacter ludwigii sp. nov., a novel Enterobacter species of clinical relevance

A new species, Enterobacter ludwigii, is presented on the basis of the characteristics of 16 strains, which were isolated from clinical specimens. These bacteria form a distinct genetic cluster in phylogenetic analyses of the population structure of the Enterobacter cloacae complex. As determined by DNA-DNA cross-hybridization experiments in microplates, this genetic cluster can be delineated from the other species of the E. cloacae complex with deltaTm values equal to or above 5 degrees C with Enterobacter hormaechei being the closest relative. The bacteria are gram-negative, fermentative, motile rods with the general characteristics of the genus Enterobacter and the E. cloacae complex in particular. E. ludwigii can be differentiated from the other Enterobacter species by its growth on myo-inositol and 3-0-methyl-D-glucopyranose. The type strain is EN-119 (= DSM 16688T = CIP 108491T).     

Cloning,expression and biochemical characterization of a β-carbonic anhydrase from the soil bacterium Enterobacter sp. B13

A recombinant carbonic anhydrase (CA, EC 4.2.1.1) from the soil-dwelling bacterium Enterobacter sp. B13 was cloned and purified by Co²⁺ affinity chromatography. Bioinformatic analysis showed that the new enzyme (denominated here B13-CA) belongs to the β-class CAs and to possess 95% homology with the ortholog enzyme from Escherichia coli encoded by the can gene, whereas its sequence homology with the other such enzyme from E. coli (encoded by the cynT gene) was of 33%. B13-CA was characterized kinetically as a catalyst for carbon dioxide hydration to bicarbonate and protons. The enzyme shows a significant catalytic activity, with the following kinetic parameters at 20?°C and pH of 8.3: k_cat of 4.8?×?10⁵?s^?1 and k_cat/K_m of 5.6?×?10⁷ M^?1?×?s^?1. This activity was potently inhibited by acetazolamide which showed a K_I of 78.9?nM. Although only this compound was investigated for the moment as B13-CA inhibitor, further studies may reveal new classes of inhibitors/activators of this enzyme which may show biomedical or environmental applications, considering the posssible role of this enzyme in CaCO₃ biomineralization processes.     

Frankia in the rhizosphere of nonhost plants: A comparison with root-associated N2-fixing Enterobacter,Klebsiella and Pseudomonas

Bacterial growth in the rhizosphere and resulting changes in plant growth parameters were studied in small aseptic seedlings of birch (Betula pendula and B. pubescens) and grasses (Poa pratensis and Festuca rubra). The seedlings were inoculated with three Frankia strains (Ai1a and Ag5b isolated from native Alnus root nodules and Ai17 from a root nodule induced by soil originating from a Betula pendula stand), and three associative N₂-fixing bacteria (Enterobacter agglomerans, Klebsiella pneumoniae and Pseudomonas sp., isolated from grass roots). Microscopic observations showed that all the Frankia strains were able to colonize and grow on the root surface of the plants tested without addition of an exogenous carbon source. No net growth of the associative N₂-fixers was observed in the rhizosphere, although inoculum viable counts were maintained over the experimental period. Changes in both the biomass and morphology of plant seedlings in response to bacterial inoculation were recorded, which were more dependent on the plant species than on the bacterial strain.     

一株霍氏肠杆菌对四环素的降解作用及其降解产物的毒性评估

【目的】抗生素作为新兴污染物,已经引起社会的极大关注。针对四环素有效降解菌株缺乏这一现状,本研究旨在筛选和鉴定具有降解四环素功能的菌株,分析其降解特性和降解作用类型、初步探讨其降解活性物质定位并评估其降解产物的生理毒性。【方法】以四环素为唯一碳源,从受四环素污染的猪场污泥中筛选四环素降解菌株;结合菌落形态学特征、生理生化特征、扫描电镜观察和16S rRNA基因测序鉴定菌株,通过不同外源碳、pH及去除动力学阐明菌株对四环素的降解特性,提取菌株不同成分探讨其去除四环素的作用类型,并进一步从细胞内液和细胞外液开展生物降解的活性物质定位,最后评估降解产物的生理毒性。【结果】筛选鉴定得到一株霍氏肠杆菌(Enterobacter hormaechei) MEH2305,pH为7.0和添加10 g/L外源碳胰蛋白胨是其发挥降解作用的最适条件。MEH2305通过非生物降解和生物降解的共同作用,在培养第7天对四环素总去除率达到68% (对土霉素和盐酸强力霉素去除率分别为53%和56%),其分泌的细胞内液和细胞外液对四环素的去除率分别为40.77%和31.18%。同时,与未经MEH2305处理的四环素对照组比较,MEH2305降解四环素的产物对革兰氏阴性大肠杆菌(Escherichia coli) K88和革兰氏阳性枯草芽孢杆菌(Bacillus subtilis) 168的生理毒性作用显著降低。【结论】MEH2305可以作为一株潜在的有效且安全的四环素降解菌株,应用于抗生素的环境治理领域。     

Kinetics and modeling of hexavalent chromium reduction in Enterobacter cloacae

Kinetics of bacterial reduction of toxic hexavalent chromium (chromate: CrO(4) (2-)) was investigated using batch and fedbatch cultures of Enterobacter cloacae strain HO1. In fedbatch cultures, the CrO(4) (2-) feed was controlled on the basis of the rate of pH change. This control strategy has proven to be useful for avoiding toxic CrO(4) (2-) overload. A simple mathematical model was developed to describe the bacterial process of CrO(4) (2-) reduction. In this model, two types of bacterial cells were considered: induced, CrO(4) (2-)-resistant cells and uninduced, sensitive ones. Only resistant cells were assumed to be able to reduce CrO(4) (2-). These fundamental ideas were supported by the model predictions which well approximated all experimental data. In a simulation study, the model was also used to optimize fed-batch cultures, instead of lengthy and expensive laboratory experiments. (c) 1993 John Wiley & Sons, Inc.