Introduction: Enterobacter cloacae strains have been isolated from Eastern Cape hospitalised patients. Methodology: We have molecularly characterised blaOXA-48-, blaIMP- and blaVIM-expressing E. cloacae isolates demon...Introduction: Enterobacter cloacae strains have been isolated from Eastern Cape hospitalised patients. Methodology: We have molecularly characterised blaOXA-48-, blaIMP- and blaVIM-expressing E. cloacae isolates demonstrating resistance to carbapenems from five hospitals by multilocus sequence typing. Organism identification and antimicrobial susceptibility testing was done using automated systems and the isolates were screened for carbapenemases using either conventional or real-time PCR and then typed using multilocus sequence typing. Further characterisation of IMP-type-producing E. cloacae isolates, an unusual occurrence in South Africa, was performed by pulsed-field gel electrophoresis. Results and Conclusion: Twenty-five E. cloacae isolates from 24 patients were investigated. Eighteen (72%) isolates harboured either one of the following genes: blaIMP, blaVIM or blaOXA-48. Multilocus sequence typing data and pulsed-field gel electrophoresis showed that several strains from the same geographical region and hospitals were genetically related.展开更多
A bacteria strain for the degradation of hydrolyzed polyacrylamide (HPAM) was isolated from a curing pot in HPAM distribution station of Daqing Oilfield using Hungate anaerobic technique.The isolate was investigated f...A bacteria strain for the degradation of hydrolyzed polyacrylamide (HPAM) was isolated from a curing pot in HPAM distribution station of Daqing Oilfield using Hungate anaerobic technique.The isolate was investigated from morphological,physiological,biochemical and molecular characterization.It is a Gram-negative,short-bacillus,non-spore-forming anaerobic bacteria with an optimum growth at 8.0 pH at 40℃.It can reduce sulfate to H2S.Alignment of 16S ribosomal DNA and 16S-23S ribosomal DNA intergenic spacer sequences suggests that this isolate is closely related to the Enterobacter cloacae.The isolate is identified as a new strain belonging to Enterobacter genus,temporarily named as Enterobacter cloacae I7.Analysis results of infrared spectroscopy (IR) show that the bacteria can use HPAM as the only carbon source,change the structure of HPAM polymer surface,and realize the hydrolysis of amide to carboxyl group by hydrolysis mechanism.It can degrade the side chain and change some functional groups,which obviously decreases the viscosity.GC-MS analysis indicates that the determined low-molecular weight degradation products of HPAM are polyacrylamide fragments with duplet bond,epoxy as well as carbonyl group,but most of them are acrylamide oligomer derivatives.展开更多
Enterobacter cloacae CYS-25 strain was isolated from a chromate plant. This bacterium was capable of resisting high hexavalent chromium concentration and reducing Cr（VI） under aerobic condition. CrO4^2- stimulated t...Enterobacter cloacae CYS-25 strain was isolated from a chromate plant. This bacterium was capable of resisting high hexavalent chromium concentration and reducing Cr（VI） under aerobic condition. CrO4^2- stimulated the increase of bacterial size and production of compact convex paths containing chromium on the bacterial surface. The increase of bacterial size was caused by integrative growth but not extracellular polymeric substance hyperplasia. IR and SDS-PAGE analyses showed the extracellular polymeric substance （EPS） components were mainly proteins and had no obvious changes whether the strains were induced by Cr（VI） or not. The EPS was amorphous and contained trivalent chromium. Under CrO4^2- growth condition, the extracellular substance of Enterobacter cloacae CYS-25 strains and Cr（VI） had redox reaction. The products were Cr^3＊-protein complexes which formed a piece of compact convex paths on the surface of bacteria and prevented Cr（VI） from entering into cells.展开更多
Fourier Transform Infrared Spectroscopy (FTIR) was used to quantify total monosaccharide content in the bacterium Enterobacter cloacae and several of its biofilm mutants. Bacterial biofilm samples were grown on trypti...Fourier Transform Infrared Spectroscopy (FTIR) was used to quantify total monosaccharide content in the bacterium Enterobacter cloacae and several of its biofilm mutants. Bacterial biofilm samples were grown on trypticase soy agar, and 30 μL aliquots of aqueous sample bacterial plus biofilm were deposited into the center of barium fluoride crystals and dried at 50°C for 1-hour before being scanned by FTIR. The total amounts of monosaccharides were estimated using the absorbance of the mono-saccharide peak, 1192 - 958 cm–1, and normalized using the amide II peak, 1585 - 1483 cm–1. This method provided a linear correlation between the absorbance of the monosaccharide peak and concentration of monosaccharide in standard monosaccharides, fructose, glucose, mannose, and rhamnose, over a concentration range of 0.5 - 2.0 mg/mL.展开更多
The nifA gene is an important regulatory gene and its product, NifA protein, regulates the expression of many nif genes involved in the nitrogen fixation process. We introduced multiple copies of the constitutively ex...The nifA gene is an important regulatory gene and its product, NifA protein, regulates the expression of many nif genes involved in the nitrogen fixation process. We introduced multiple copies of the constitutively expressed Sinorhizobium meliloti (Sm) or Enterobacter cloacae (Ec) nifA gene into both the nifA mutant strain SmY and the wild-type strain Sm1021. Root nodules produced by SmY containing a constitutively expressed Sm nifA gene were capable of fixing nitrogen, while nodules produced by SmY containing the Ec nifA gene remained unable to fix nitrogen, as is the case for SmY itself. However, transfer of an additional Sm nifA gene into Sm1021 improved the nitrogen-fixing efficiency of root nodules to a greater extent than that observed upon transfer of the Ec nifA gene into Sm1021. Comparative analysis of amino acid sequences between Sm NifA and Ec NifA showed that the N-terminal domain was the least similar, but this domain is indispensable for complementation of the Fix? phenotype of SmY by Sm NifA. We conclude that more than one domain is involved in determining functional differences between Sm NifA and Ec NifA.展开更多
Expression of the nitrogen fixation (nif ) genes is tightly regulated by two proteins NifA and NifL in the (-subdivision of the proteobacteria. NifA is a transcriptional activator, which can be inactivated by NifL in ...Expression of the nitrogen fixation (nif ) genes is tightly regulated by two proteins NifA and NifL in the (-subdivision of the proteobacteria. NifA is a transcriptional activator, which can be inactivated by NifL in the presence of oxygen or excess fixed nitrogen. A direct interaction between E. cloacae NifL and NifA was detected using the bacterial two-hybrid system. This interaction was accelerated in the presence of fixed nitrogen, while oxygen had no effect. NifL proteins, with their C-terminus being deleted, completely lost the ability to interact with NifA. The data suggest that the C-terminal domain of NifL acts as a sensor of the nitrogen status of the cell and mediates interaction with NifA.展开更多
The NifA protein is the central regulator of the nitrogen fixation genes. It activates transcription of nif genes by an alternative holoenzyme form of RNA polymerase containing the σ54 factor. The NifA protein from K...The NifA protein is the central regulator of the nitrogen fixation genes. It activates transcription of nif genes by an alternative holoenzyme form of RNA polymerase containing the σ54 factor. The NifA protein from Klebsiella pneumoniae consists of the N-terminal domain of unknown function, the central catalytic domain with ATPase activity and the C-terminal DNA-binding domain. The Kp NifA protein is sensitive to temperature, while the Enterobacter cloacae NifA protein is less sensitive to temperature than Kp NifA. Our results show that the N-terminal domain of NifA plays the decisive role in the temperature sensitivity of the protein.展开更多
Objective To investigate the status of the drug resistance and the ampC gene expression of Enterobacter cloacae . Methods Disk diffusion tests were made for detecting the susceptibility of antimicrobial agents agai...Objective To investigate the status of the drug resistance and the ampC gene expression of Enterobacter cloacae . Methods Disk diffusion tests were made for detecting the susceptibility of antimicrobial agents against Enterobacter cloacae . AmpC gene was amplified by polymerase chain reaction (PCR) and verified by DNA sequencing. AmpC gene expression was analyzed according to antimicrobial agent sensitive phenotype. Results The sensitivity rates of 144 strains to imipenam,cefepime and cefoperazone/sulbactam were 98.61%,65.97% and 63.89%,respectively. The sensitivity rates of 144 strains to other antimicrobial agents were lower. Among the 144 strains 120 were found to be positive by PCR for ampC. The PCR product showed high homology to the GenBank ampC sequence. Stably derepressed strains,hyperinducible strains and unexpressing or lower level expressing strains accounted for 30.0% (36/120),37.5% (45/120),and 32.5% (39/120),respectively. Fifty-six out of 120 strains (46.67%) also produced extended spectrum β-lactamases (ESBLs). The hyperinducible strains were highly sensitive to all the antimicrobial agents except amoxicillin/clavulanic acid and cefuroxime,while the stably derepressed strains were only sensitive to imipenam and cefepime. However,sensitivity to cefepime decreased if the strains also produced ESBLs. Conclusions The durg resistant status of Enterobacter cloacae is severe. Clearing out the expressive status of ampC gene will be helpful in selection of antimicrobial agents in the treatment of clinical infection.展开更多
To understand the antagonistic mechanism of the broad spectrum antagonistic Enterobacter cloacae B8,Tn5 transposon-mediated mutagenesis is performed using suicide plasmid pZJ25. Two mutant strains that lost antagonist...To understand the antagonistic mechanism of the broad spectrum antagonistic Enterobacter cloacae B8,Tn5 transposon-mediated mutagenesis is performed using suicide plasmid pZJ25. Two mutant strains that lost antagonistic character are isolated. Tagging with kanr gene on Tn5,an antagonistic related DNA fragment, the F fragment, right of the Tn5 insertion site is cloned in a plasmid named pTLF,from one of the mutant strains B8F. The 733 bp F fragment is then sequenced after subcloning. Genomic DNA of the original B8 strain is isolated, digested with Pst I and ligated to Pst I cassette. DNA fragments left and right of the F fragment are amplified from the Pst I cassette library using cassette primer and specific primers designed according to known sequence. 1106 bp sequence left of the F fragment and 664bp sequence right of the F fragment are finally obtained. Bioinformatics analysis shows that the contig assembled from the sequences of the cloned antagonistic related DNA fragments of B8 encodes three ORFs and is homogeneous to admM,admN and admO genes of Pantoea agglomerans andrimid biosynthetic gene cluster (AY192157). The ORF, named anrF gene which encodes a polyketide synthase, knocked out by Tn5 insertion, is a homology of admM and the insertion site of Tn5 is at 214 bp upstream of the stop codon. It is concluded that the anrF gene is a gene related to the antagonistic activity of E. cloacae B8, and speculated that the antagonistic substance produced by B8 is an andrimid.展开更多
In order to provide useful information for effective control and clinical therapy of infection,the resistance status and the rate of carrying AmpC β-lactamase of Enterobacter cloacae(E.cloacae)were investigated.By VI...In order to provide useful information for effective control and clinical therapy of infection,the resistance status and the rate of carrying AmpC β-lactamase of Enterobacter cloacae(E.cloacae)were investigated.By VITEK(Bacterial automatic biochemical analyzer),the isolates of E.cloacae were identified and the drug resistance was measured.The AmpC enzyme was detected by thefive-disk diffusion test.Antibiotic sensitivity test showed that the resistance effects of E.cloacae to cefazolin,cefoxitin and ampicillin were more serious,with resistant rates of 80.5%,75.3%and 70.1%,respectively.However,it was more sensitive to Sulperazone(cefoperazone/sulbactam,13.0%),amikacin(16.9%)and ciprofloxacin(19.5%).Meanwhile,the phenotype detection showed that 35.06%(27/77)isolates of E.cloacae produced AmpCβ-lactamase.Most of E.cloacae are multi-drug resistant strains.Sulperazone(cefoperazone/sulbactam),a kind of componentβ-lactamase,is a more effective antibiotic for treating infection caused by E.cloacae.Unreasonable application of the third generation cephalosporins plays an important role in leading to emergence of high-yield AmpCβ-lactamase strains,so antibiotics should be used wisely.展开更多
文摘Introduction: Enterobacter cloacae strains have been isolated from Eastern Cape hospitalised patients. Methodology: We have molecularly characterised blaOXA-48-, blaIMP- and blaVIM-expressing E. cloacae isolates demonstrating resistance to carbapenems from five hospitals by multilocus sequence typing. Organism identification and antimicrobial susceptibility testing was done using automated systems and the isolates were screened for carbapenemases using either conventional or real-time PCR and then typed using multilocus sequence typing. Further characterisation of IMP-type-producing E. cloacae isolates, an unusual occurrence in South Africa, was performed by pulsed-field gel electrophoresis. Results and Conclusion: Twenty-five E. cloacae isolates from 24 patients were investigated. Eighteen (72%) isolates harboured either one of the following genes: blaIMP, blaVIM or blaOXA-48. Multilocus sequence typing data and pulsed-field gel electrophoresis showed that several strains from the same geographical region and hospitals were genetically related.
基金Sponsored by the Country from Branch Fund Significant International Cooperation Item(Grant No.50521140075)
文摘A bacteria strain for the degradation of hydrolyzed polyacrylamide (HPAM) was isolated from a curing pot in HPAM distribution station of Daqing Oilfield using Hungate anaerobic technique.The isolate was investigated from morphological,physiological,biochemical and molecular characterization.It is a Gram-negative,short-bacillus,non-spore-forming anaerobic bacteria with an optimum growth at 8.0 pH at 40℃.It can reduce sulfate to H2S.Alignment of 16S ribosomal DNA and 16S-23S ribosomal DNA intergenic spacer sequences suggests that this isolate is closely related to the Enterobacter cloacae.The isolate is identified as a new strain belonging to Enterobacter genus,temporarily named as Enterobacter cloacae I7.Analysis results of infrared spectroscopy (IR) show that the bacteria can use HPAM as the only carbon source,change the structure of HPAM polymer surface,and realize the hydrolysis of amide to carboxyl group by hydrolysis mechanism.It can degrade the side chain and change some functional groups,which obviously decreases the viscosity.GC-MS analysis indicates that the determined low-molecular weight degradation products of HPAM are polyacrylamide fragments with duplet bond,epoxy as well as carbonyl group,but most of them are acrylamide oligomer derivatives.
基金This work was supported by the National Natural Science Foundation of China (20501020, 40772034) Nanoscience Foundation of China (90406024)+2 种基金Natural Science Foundation of Fujian Province (No. X0650094/2006J0383)973 program (2007CB815601) the Special Project on Science and Technology of Fujian Province (2005YZ1026)
文摘Enterobacter cloacae CYS-25 strain was isolated from a chromate plant. This bacterium was capable of resisting high hexavalent chromium concentration and reducing Cr（VI） under aerobic condition. CrO4^2- stimulated the increase of bacterial size and production of compact convex paths containing chromium on the bacterial surface. The increase of bacterial size was caused by integrative growth but not extracellular polymeric substance hyperplasia. IR and SDS-PAGE analyses showed the extracellular polymeric substance （EPS） components were mainly proteins and had no obvious changes whether the strains were induced by Cr（VI） or not. The EPS was amorphous and contained trivalent chromium. Under CrO4^2- growth condition, the extracellular substance of Enterobacter cloacae CYS-25 strains and Cr（VI） had redox reaction. The products were Cr^3＊-protein complexes which formed a piece of compact convex paths on the surface of bacteria and prevented Cr（VI） from entering into cells.
文摘Fourier Transform Infrared Spectroscopy (FTIR) was used to quantify total monosaccharide content in the bacterium Enterobacter cloacae and several of its biofilm mutants. Bacterial biofilm samples were grown on trypticase soy agar, and 30 μL aliquots of aqueous sample bacterial plus biofilm were deposited into the center of barium fluoride crystals and dried at 50°C for 1-hour before being scanned by FTIR. The total amounts of monosaccharides were estimated using the absorbance of the mono-saccharide peak, 1192 - 958 cm–1, and normalized using the amide II peak, 1585 - 1483 cm–1. This method provided a linear correlation between the absorbance of the monosaccharide peak and concentration of monosaccharide in standard monosaccharides, fructose, glucose, mannose, and rhamnose, over a concentration range of 0.5 - 2.0 mg/mL.
文摘The nifA gene is an important regulatory gene and its product, NifA protein, regulates the expression of many nif genes involved in the nitrogen fixation process. We introduced multiple copies of the constitutively expressed Sinorhizobium meliloti (Sm) or Enterobacter cloacae (Ec) nifA gene into both the nifA mutant strain SmY and the wild-type strain Sm1021. Root nodules produced by SmY containing a constitutively expressed Sm nifA gene were capable of fixing nitrogen, while nodules produced by SmY containing the Ec nifA gene remained unable to fix nitrogen, as is the case for SmY itself. However, transfer of an additional Sm nifA gene into Sm1021 improved the nitrogen-fixing efficiency of root nodules to a greater extent than that observed upon transfer of the Ec nifA gene into Sm1021. Comparative analysis of amino acid sequences between Sm NifA and Ec NifA showed that the N-terminal domain was the least similar, but this domain is indispensable for complementation of the Fix? phenotype of SmY by Sm NifA. We conclude that more than one domain is involved in determining functional differences between Sm NifA and Ec NifA.
基金This work was supported by a grant in aid of the Shanghai Institutes of Biological Sciences, the Chinese Academy of Sciences "973" Project of the Ministry of Science and Technology, China (Grant No. 2001CB108901)
文摘Expression of the nitrogen fixation (nif ) genes is tightly regulated by two proteins NifA and NifL in the (-subdivision of the proteobacteria. NifA is a transcriptional activator, which can be inactivated by NifL in the presence of oxygen or excess fixed nitrogen. A direct interaction between E. cloacae NifL and NifA was detected using the bacterial two-hybrid system. This interaction was accelerated in the presence of fixed nitrogen, while oxygen had no effect. NifL proteins, with their C-terminus being deleted, completely lost the ability to interact with NifA. The data suggest that the C-terminal domain of NifL acts as a sensor of the nitrogen status of the cell and mediates interaction with NifA.
文摘The NifA protein is the central regulator of the nitrogen fixation genes. It activates transcription of nif genes by an alternative holoenzyme form of RNA polymerase containing the σ54 factor. The NifA protein from Klebsiella pneumoniae consists of the N-terminal domain of unknown function, the central catalytic domain with ATPase activity and the C-terminal DNA-binding domain. The Kp NifA protein is sensitive to temperature, while the Enterobacter cloacae NifA protein is less sensitive to temperature than Kp NifA. Our results show that the N-terminal domain of NifA plays the decisive role in the temperature sensitivity of the protein.
文摘Objective To investigate the status of the drug resistance and the ampC gene expression of Enterobacter cloacae . Methods Disk diffusion tests were made for detecting the susceptibility of antimicrobial agents against Enterobacter cloacae . AmpC gene was amplified by polymerase chain reaction (PCR) and verified by DNA sequencing. AmpC gene expression was analyzed according to antimicrobial agent sensitive phenotype. Results The sensitivity rates of 144 strains to imipenam,cefepime and cefoperazone/sulbactam were 98.61%,65.97% and 63.89%,respectively. The sensitivity rates of 144 strains to other antimicrobial agents were lower. Among the 144 strains 120 were found to be positive by PCR for ampC. The PCR product showed high homology to the GenBank ampC sequence. Stably derepressed strains,hyperinducible strains and unexpressing or lower level expressing strains accounted for 30.0% (36/120),37.5% (45/120),and 32.5% (39/120),respectively. Fifty-six out of 120 strains (46.67%) also produced extended spectrum β-lactamases (ESBLs). The hyperinducible strains were highly sensitive to all the antimicrobial agents except amoxicillin/clavulanic acid and cefuroxime,while the stably derepressed strains were only sensitive to imipenam and cefepime. However,sensitivity to cefepime decreased if the strains also produced ESBLs. Conclusions The durg resistant status of Enterobacter cloacae is severe. Clearing out the expressive status of ampC gene will be helpful in selection of antimicrobial agents in the treatment of clinical infection.
文摘To understand the antagonistic mechanism of the broad spectrum antagonistic Enterobacter cloacae B8,Tn5 transposon-mediated mutagenesis is performed using suicide plasmid pZJ25. Two mutant strains that lost antagonistic character are isolated. Tagging with kanr gene on Tn5,an antagonistic related DNA fragment, the F fragment, right of the Tn5 insertion site is cloned in a plasmid named pTLF,from one of the mutant strains B8F. The 733 bp F fragment is then sequenced after subcloning. Genomic DNA of the original B8 strain is isolated, digested with Pst I and ligated to Pst I cassette. DNA fragments left and right of the F fragment are amplified from the Pst I cassette library using cassette primer and specific primers designed according to known sequence. 1106 bp sequence left of the F fragment and 664bp sequence right of the F fragment are finally obtained. Bioinformatics analysis shows that the contig assembled from the sequences of the cloned antagonistic related DNA fragments of B8 encodes three ORFs and is homogeneous to admM,admN and admO genes of Pantoea agglomerans andrimid biosynthetic gene cluster (AY192157). The ORF, named anrF gene which encodes a polyketide synthase, knocked out by Tn5 insertion, is a homology of admM and the insertion site of Tn5 is at 214 bp upstream of the stop codon. It is concluded that the anrF gene is a gene related to the antagonistic activity of E. cloacae B8, and speculated that the antagonistic substance produced by B8 is an andrimid.
基金supported by the Key Subject of Tianjin Medical University(No.2004xk47).
文摘In order to provide useful information for effective control and clinical therapy of infection,the resistance status and the rate of carrying AmpC β-lactamase of Enterobacter cloacae(E.cloacae)were investigated.By VITEK(Bacterial automatic biochemical analyzer),the isolates of E.cloacae were identified and the drug resistance was measured.The AmpC enzyme was detected by thefive-disk diffusion test.Antibiotic sensitivity test showed that the resistance effects of E.cloacae to cefazolin,cefoxitin and ampicillin were more serious,with resistant rates of 80.5%,75.3%and 70.1%,respectively.However,it was more sensitive to Sulperazone(cefoperazone/sulbactam,13.0%),amikacin(16.9%)and ciprofloxacin(19.5%).Meanwhile,the phenotype detection showed that 35.06%(27/77)isolates of E.cloacae produced AmpCβ-lactamase.Most of E.cloacae are multi-drug resistant strains.Sulperazone(cefoperazone/sulbactam),a kind of componentβ-lactamase,is a more effective antibiotic for treating infection caused by E.cloacae.Unreasonable application of the third generation cephalosporins plays an important role in leading to emergence of high-yield AmpCβ-lactamase strains,so antibiotics should be used wisely.
