水稻根际兼性厌氧催娩克氏杆菌和阴沟肠杆菌的固氮与氢代谢

兼性厌氧细菌Enterobacter cloacae菌株E-26和Klebsiella oxytoca菌株NG-13的氢酶与固氮酶同时形成。固氮的最佳碳源为蔗糖、葡萄糖和丙酮酸,此外延胡索酸和苹果酸也能支持固氮。支持固氮的碳源也支持放氢,两者动力学基本一致。40％乙炔预处理后,吸氢活性下跌,放氢量未增加;NH_4~ 抑制固氮酶,但未导致放氢量降低;可能E-26菌株的放氢主要依赖于氢酶。菌株E-26和NG-13的吸氢反应,既能以O_2为电子受体,也能以延胡索酸、硝酸、MB为电子受体。但仅延胡索酸为电子受体时,E-26菌的固氮活性被分子H_2促进,它的氢吸收利用与固氮相偶联;而在CO_2和NH_4~ 代谢与H_2利用之间并无明显相关性,吸氢活性不被CO_2和NH_4~ 促进。     

皂角发酵物对红托竹荪病害防治及土壤主要微生物群落影响

为了研究皂角发酵物对贵州当地特色产业红托竹荪的病害防治及促生长作用,本研究在室内分离病原菌并进行平板对峙实验,田间试验设计4个处理：不施用药剂的常规处理、解淀粉芽孢杆菌HN11菌液、皂角粉末、皂角发酵物,调查防治效果,测量菌蛋大小及个数,检测土壤微生物群落变化。从发病组织中分离出一株病原菌,鉴定为阴沟肠杆菌Enterobacter cloacae。皂角发酵物对竹荪病害田间防治效果达77.86%,生长面积提高61.22%。土壤微生物群落中细菌和真菌分析结果显示,相较于其他处理组,皂角发酵物处理组中竹荪相对丰度占比最大,达25.83%。皂角发酵物能有效防治红托竹荪病害,促进红托竹荪菌蛋生长,减少化学农药的用量,促进生态循环,保障农产品食用安全,提升皂角和竹荪产业综合效益。     

Survival and growth of Enterobacter sakazakii in infant rice cereal reconstituted with water, milk, liquid infant formula, or apple juice

AIMS: To determine survival and growth characteristics of Enterobacter sakazakii in infant rice cereal as affected by type of liquid used for reconstitution and storage temperature after reconstitution. METHODS AND RESULTS: A commercially manufactured dry infant rice cereal was reconstituted with water, apple juice, milk, or liquid infant formula, inoculated with a 10-strain mixture of E. sakazakii at populations of 0.27, 0.93, and 9.3 CFU ml(-1), and incubated at 4, 12, 21 or 30 degrees C for up to 72 h. Growth did not occur in cereal reconstituted with apple juice, regardless of storage temperature, or in cereal reconstituted with water, milk, or formula and stored at 4 degrees C. The lag time for growth in cereal reconstituted with water, milk, or formula was decreased as the incubation temperature (12, 21 and 30 degrees C) was increased. Upon reaching maximum populations of 7-8 log10 CFU ml(-1), in some instances populations decreased to nondetectable levels during subsequent storage which was concurrent with decreases in pH. CONCLUSIONS: Enterobacter sakazakii initially at very low populations can rapidly grow in infant rice cereal reconstituted with water, milk, or infant formula. SIGNIFICANCE AND IMPACT OF THE STUDY: Reconstituted infant rice cereal can support luxuriant growth of E. sakazakii. Reconstituted cereal that is not immediately consumed should be discarded or stored at a temperature at which E. sakazakii and other food-borne pathogens cannot grow.     

Development of a fast DNA-DNA hybridization method based on melting profiles in microplates

DNA-DNA hybridization is still the “gold standard” for the genotypic delineation of bacterial species. However, it is not widely used because traditional DNA-DNA hybridization techniques are rather time-consuming and not easy to perform in routine laboratories. In the present study, DNA of reference strains was digested with Sau3A, ligated with linker oligonucleotides S1/2 and in vitro amplified. The amplified DNA fragments were immobilized on MaxiSorb 96-well plates. DNA isolated from target strains was also digested with Sau3A, ligated with linker oligonuleotides P1/2 and in vitro amplified in the presence of digoxygenin modified dUTP. The labeled amplificate was hybridized to the immobilized reference DNA under isothermal conditions. Thermal denaturation curves of the DNA-DNA hybrids were obtained by using washing solutions of increasing stringency. Remaining hybrids were colorimetrically detected with anti-digoxygenin-horseradish peroxidase anti-bodies. The new method was validated with strains of the genus Pedioccocus for which DNA-DNA similarities have also been determined by the filter hybridization method. In addition, DNA-DNA hybridizations were performed with genotypically defined Enterobacter species.     

Polymerase chain reaction amplification length-dependent ethidium monoazide suppression power for heat-killed cells of Enterobacteriaceae

The polymerase chain reaction (PCR) can confirm the presence of bacteria, but it is unable to differentiate between live and dead bacteria. Although ethidium monoazide (EMA)- and propidium monoazide (PMA)-based PCR have been evaluated, a quantity of ≥ 10(3)cells/ml dead cells produces a false-positive reading at 40 to 50 cycles (K. Rudi et al., Appl. Environ. Microbiol. 71 (2005) 1018-1024). After confirming the precision of real-time PCR of a long DNA target (16S or 23S ribosomal RNA [rRNA] gene, 1490 or 2840 bp), we evaluated the degree of suppression of an EMA treatment on the 16S/23S PCR using various amplification lengths (110-2840 bp) with heat-killed cells of Enterobacteriaceae (e.g., Salmonella enteritidis). We found that the inhibition rate was proportional to the PCR amplification length; short DNA (110 bp) amplification slightly delayed the threshold cycle (C(T)) of heat-killed cells of Enterobacteriaceae when compared with no EMA treatment. Regardless of the amplification length, the C(T) delay using live cells of Enterobacteriaceae with EMA was negligible. Thus, our real-time PCR of a long DNA (16S or 23S) template following EMA treatment is a rapid viable bacterial assay, which can potentially target all genera, for testing pasteurized milk that may have originally been contaminated with high levels of dead bacteria.     

Amelioration of phytotoxic effects of Cd on mung bean seedlings by gluconic acid secreting rhizobacterium <Emphasis Type="Italic">Enterobacter asburiae</Emphasis> PSI3 and implication of role of organic acid

Mung bean seedlings inoculated with Enterobacter asburiae PSI3, a gluconic acid-producing rhizosphere isolate, enhanced plant growth in the presence of phytotoxic levels of Cd²⁺ in gnotobiotic pot experiments as compared to the uninoculated Cd-treated plants. Addition of organic acids to Cd-stressed seedlings promoted root elongation. Hematoxylin competition assays showed that organic acids could displace Cd²⁺ from the Cd²⁺: hematoxylin complex in the same order of effectiveness as was found for restoration of root net elongation viz. oxalate > malate > succinate while gluconate was effective at higher concentrations. Root associated Cd²⁺, assessed by hematoxylin staining of roots was found to be reduced when roots were treated with organic acid. Cd stress increased antioxidant enzymes such as peroxidase and superoxide dismutase in mung bean roots while organic acid treatment suppressed the up-regulation of these enzymes by Cd.     

Attachment,colonization and proliferation ofAzospirillum brasilense andEnterobacter spp. on root surface of grasses

Root colonization studies, employing immunofluorescence and using locally isolated strains, showed thatEnterbacter sp. QH7 andEnterobacter agglomerans AX12 attached more readily to the roots of most plants compared withAzospirillum brasilense JM82. Heat treatment of either root or inoculum significantly decreased the adsorption of bacteria to the root surface. Kallar grass and rice root exudates sustained the growth ofA. brasilense JM82,Enterobacter sp. QH7 andE. agglomerans AX12 in Hoagland and Fahraeus medium. All the strains colonized kallar grass and rice roots in an axenic culture system. However, in studies involving mixed cultures,A. brasilense JM82 was inhibited byEnterobacter sp. QH7 in kallar grass rhizosphere and the simultaneous presence ofEnterobacter sp. QH7 andE. agglomerans AX12 suppressed the growth ofA. brasilense JM82 in rice rhizosphere. The bacterial colonization pattern changed from dispersed to aggregated within 3 days of inoculation. The colonization sites corresponded mainly to the areas where root mucigel was present. The area around the point of emergence of lateral roots usually showed maximum colonization.     

Cloning and sequencing of the beta-lactamase gene and surrounding DNA sequences of Citrobacter braakii,Citrobacter murliniae,Citrobacter werkmanii,Escherichia fergusonii and Enterobacter cancerogenus

To further identify the origins of plasmid-mediated cephalosporinases that are currently spreading worldwide, the chromosomal beta-lactamase genes of Citrobacter braakii, Citrobacter murliniae, Citrobacter werkmanii reference strains and of Escherichia fergusonii and Enterobacter cancerogenus clinical isolates were cloned and expressed into Escherichia coli and sequenced. These beta-lactamases had all a single pI value >8 and conferred a typical AmpC-type resistance pattern in E. coli recombinant strains. The cloned inserts obtained from genomic DNAs of each strain encoded Ambler class C beta-lactamases. The AmpC-type enzymes of C. murliniae, C. braakii and C. werkmanii shared 99%, 96% and 95% amino acid sequence identity, respectively, with chromosomal AmpC beta-lactamases from Citrobacter freundii. The AmpC-type enzyme of E. cancerogenus shared 85% amino acid sequence identity with the chromosomal AmpC beta-lactamase of Enterobacter cloacae OUDhyp and the AmpC-type enzyme of E. fergusonii shared 96% amino acid sequence identity with that of E. coli K12. The ampC genes, except for E. fergusonii, were associated with genes homologous to regulatory ampR genes of other chromosomal class C beta-lactamases that explain inducibility of beta-lactamase expression in these strains. This work provides further evidence of the molecular heterogeneity of class C beta-lactamases.     

Molecular characterization of the alpha-glucosidase activity in Enterobacter sakazakii reveals the presence of a putative gene cluster for palatinose metabolism

Enterobacter sakazakii is considered an opportunistic pathogen for premature infants and neonates. Although E. sakazakii has been isolated from various types of food, recontaminated dried infant formula has been epidemiologically identified as the major source of infection. Amongst others, alpha-glucosidase activity is one of the most important biochemical features, which differentiates E. sakazakii from other species in the family Enterobacteriaceae and has therefore been used as a selective marker in the development of differential media. However, it has been shown, that methods based on this biochemical feature are prone to producing false-positive results for presumptive E. sakazakii colonies due to the presence of this enzymatic activity in other species of the Enterobacteriaceae. Therefore, elucidation of the molecular basis responsible for the biochemical feature in E. sakazakii would provide novel targets suitable for the development of more specific and direct identification systems for this organism. By applying the bacterial artificial chromosome (BAC) approach, along with heterologous gene expression in Escherichia coli, the molecular basis of the alpha-glucosidase activity in E. sakazakii was characterized. Here we report the identification of two different alpha-glucosidase encoding genes. Homology searches of the deduced amino acid sequences revealed that the proteins belong to a cluster of gene products putatively responsible for the metabolism of isomaltulose (palatinose; 6-O-alpha-d-glucopyranosyl-d-fructose). The glycosyl-hydrolyzing activity of each protein was demonstrated by subcloning the respective open reading frames and screening of E. coli transformants for their ability to hydrolyze 4-methyl-umbelliferyl-alpha-d-glucoside. Analysis at the protein level revealed that both enzymes belong to the intracellular fraction of cell proteins. The presence of the postulated palatinose metabolism was proven by growth experiments using this sugar as a sole carbon source.     

Occurrence of Cronobacter spp. in retail foods

Aims: To study the occurrence of Cronobacter spp. in foods and to investigate the phenotypic properties of the strains isolated. Methods and Results: A total of 53 strains of Cronobacter spp. isolated from 399 food samples were identified using conventional biochemical methods and MALDI‐TOF mass spectrometry. Foods of plant origin were the most frequently contaminated samples. No Cronobacter spp. were found in infant milk formula, wheat‐based infant food, pasteurized and raw cow milk, mincemeat, chicken, chickpea and potato dumpling powder. The individual species were identified as Cronobacter sakazakii (54·7%), Cronobacter malonaticus (28·4%), Cronobacter dublinensis (7·5%), Cronobacter muytjensii (7·5%) and Cronobacter turicensis (1·9%). Cronobacter sakazakii and C. malonaticus belong to biotype 1, 2, 2a, 3, 4 and 5, 5a, respectively. Cronobacter dublinensis strains were subdivided into biotypes 6 and 12. All strains were resistant to erythromycin and two of them were resistant to both erythromycin and tetracycline. Conclusions: Cronobacter spp. were isolated from various food samples pre‐eminently of plant origin and dried food ingredients. Significance and Impact of the Study: These findings will increase and detail our knowledge of the presence and diversity of Cronobacter spp. in foods.