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Comparative Whole-Genome Analysis of Virulent and Avirulent Strains of Porphyromonas gingivalis. Tsute Chen, 2004.We used Porphyromonas gingivalis gene microarrays to compare the total gene contents of the virulent strain W83 and the avirulent type strain, ATCC 33277 . Signal ratios and scatter plots indicated that the chromosomes were very similar, with approximately 93% of the predicted genes in common, while at least 7% of them showed very low or no signals in ATCC 33277 . Verification of the array results by PCR indicated that several of the disparate genes were either absent from or variant in ATCC 33277 . Divergent features included already reported insertion sequences and ragB, as well as additional hypothetical and functionally assigned genes . Several of the latter were organized in a putative operon in W83 and encoded enzymes involved in capsular polysaccharide synthesis . Another cluster was associated with two paralogous regions of the chromosome with a low G+C content, at 41%, compared to that of the whole genome, at 48% . These regions also contained conserved and species-specific hypothetical genes, transposons, insertion sequences, and integrases and were located adjacent to tRNA genes; thus, they had several characteristics of pathogenicity islands . While this global comparative analysis showed the close relationship between W83 and ATCC 33277, the clustering of genes that are present in W83 but divergent in or absent from ATCC 33277 is suggestive of chromosomal islands that may have been acquired by lateral gene transfer . Rabbit Model of Candida albicans Biofilm Infection: Liposomal Amphotericin B Antifungal Lock Therapy. Matthew K. Schinabeck, 2004.Catheter-related infections due to Candida albicans biofilms are a leading cause of fungal nosocomial bloodstream infection . In this paper, we describe the development of a model of catheter-associated infection with C . albicans biofilms and show that antifungal lock therapy with liposomal amphotericin B is an effective treatment strategy for these infections . Silicone catheters surgically placed in New Zealand White rabbits were infected with C . albicans, and the rabbits were randomized into three groups: (i) untreated controls, (ii) liposomal amphotericin B lock, and (iii) fluconazole lock . Upon completion of therapy, blood cultures were obtained and the catheters were removed for quantitative culture and scanning electron microscopic analyses . Quantitative cultures revealed that catheters treated with liposomal amphotericin B yielded 0 CFU, which was significant compared to the untreated controls (P < 0.001) and the fluconazole-treated group (P = 0.0079) . Although fluconazole treatment tended to have lower CFU compared to untreated controls, there was no difference in mean colony counts between these two groups (1.128 ± 0.764 and 1.841 ± 1.141 log10 CFU/catheter segment, respectively; P = 0.297) . Scanning electron microscopy revealed abundant biofilm in the control and fluconazole groups, while the liposomal amphotericin B group was virtually cleared . These findings suggest a possible treatment strategy for the successful salvage of catheters infected with C . albicans biofilms and describe an animal model that may play an important role in the further study of C . albicans biofilm pathogenesis and evaluation of potential antibiofilm agents . Comparative Proteomic Analysis of Extracellular Proteins of Enterohemorrhagic and Enteropathogenic Escherichia coli Strains and Their ihf and ler Mutants. M. Li, 2004.Enterohemorrhagic and enteropathogenic Escherichia coli (EHEC and EPEC, respectively) strains are closely related human pathogens that are responsible for food-borne epidemics in many countries . Integration host factor (IHF) and the locus of enterocyte effacement-encoded regulator (Ler) are needed for the expression of virulence genes in EHEC and EPEC, including the elicitation of actin rearrangements for attaching and effacing lesions . We applied a proteomic approach, using two-dimensional polyacrylamide gel electrophoresis in combination with matrix-assisted laser desorption ionization-time of flight mass spectrometry and a protein database search, to analyze the extracellular protein profiles of EHEC EDL933, EPEC E2348/69, and their ihf and ler mutants . Fifty-nine major protein spots from the extracellular proteomes were identified, including six proteins of unknown function . Twenty-six of them were conserved between EHEC EDL933 and EPEC E2348/69, while some of them were strain-specific proteins . Four common extracellular proteins (EspA, EspB, EspD, and Tir) were regulated by both IHF and Ler in EHEC EDL933 and EPEC E2348/69 . TagA in EHEC EDL933 and EspC and EspF in EPEC E2348/69 were present in the wild-type strains but absent from their respective ler and ihf mutants, while FliC was overexpressed in the ihf mutant of EPEC E2348/69 . Two dominant forms of EspB were found in EHEC EDL933 and EPEC E2348/69, but the significance of this is unknown . These results show that proteomics is a powerful platform technology for accelerating the understanding of EPEC and EHEC pathogenesis and identifying markers for laboratory diagnoses of these pathogens . Complete Nucleotide Sequence and Genetic Organization of the 210-Kilobase Linear Plasmid of Rhodococcus erythropolis BD2. Christiane Stecker, 2003.The complete nucleotide sequence of the linear plasmid pBD2 from Rhodococcus erythropolis BD2 comprises 210,205 bp . Sequence analyses of pBD2 revealed 212 putative open reading frames (ORFs), 97 of which had an annotatable function . These ORFs could be assigned to six functional groups: plasmid replication and maintenance, transport and metalloresistance, catabolism, transposition, regulation, and protein modification . Many of the transposon-related sequences were found to flank the isopropylbenzene pathway genes . This finding together with the significant sequence similarities of the ipb genes to genes of the linear plasmid-encoded biphenyl pathway in other rhodococci suggests that the ipb genes were acquired via transposition events and subsequently distributed among the rhodococci via horizontal transfer . Identification of a High-Affinity Phosphate Transporter Gene in a Prasinophyte Alga, Tetraselmis chui, and Its Expression under Nutrient Limitation. Chih-Ching Chung, 2003.A high-affinity phosphate transporter gene, TcPHO, was isolated from a growth-dependent subtracted cDNA library of the marine unicellular alga Tetraselmis chui . The full-length cDNA of TcPHO obtained by 5' and 3' rapid amplification of cDNA ends was 1,993 bp long and encoded an open reading frame consisting of 610 amino acids . The deduced amino acid sequence of TcPHO exhibited 51.6 and 49.8% similarity to the amino acid sequences of PHO89 from Saccharomyces cerevisiae and PHO4 from Neurospora crassa, respectively . In addition, hydrophobicity and secondary structure analyses revealed 12 conserved transmembrane domains that were the same as those found in PHO89 and PHO4 . The expression of TcPHO mRNA was dependent on phosphate availability . With a low-phosphate treatment, the TcPHO mRNA concentration increased sharply to 2.72 fmol µg of total RNA-1 from day 1 to day 2 and remained at this high level from days 2 to 4 . Furthermore, rescue treatment with either phosphate or p-nitrophenyl phosphate effectively inhibited TcPHO mRNA expression . In contrast, TcPHO mRNA expression stayed at a low level (range, 0.25 to 0.28 fmol µg of total RNA-1) under low-nitrate conditions . The expression pattern suggests that TcPHO can be used as a molecular probe for monitoring phosphorus stress in T . chui . Reduction of Norwalk Virus, Poliovirus 1, and Bacteriophage MS2 by Ozone Disinfection of Water. Gwy-Am Shin, 2003.Norwalk virus and other human caliciviruses (noroviruses) are major agents of gastroenteritis, and water is a major route of their transmission . In an effort to control Norwalk virus in drinking water, Norwalk virus reduction by bench-scale ozone disinfection was determined using quantitative reverse transcription (RT)-PCR for virus assays . Two other enteric viruses, poliovirus 1 and coliphage MS2, were included for comparison, and their reductions were assayed by infectivity assays as well as by RT-PCR . Virus reductions by ozone were determined using a dose of 0.37 mg of ozone/liter at pH 7 and 5°C for up to 5 min . Based on two RT-PCR assays, the reductions of Norwalk virus were >3 log10 within a contact time of 10 s, and these were similar to the reductions of the other two viruses determined by the same assay methods . Also, the virus reductions detected by RT-PCR assays were similar to those detected by infectivity assays, indicating that the RT-PCR assay is a reliable surrogate assay for both culturable and nonculturable viruses disinfected with ozone . Overall, the results of this study indicate that Norwalk virus as well as other enteric viruses can be reduced rapidly and extensively by ozone disinfection and that RT-PCR is a useful surrogate assay for both culturable and nonculturable viruses disinfected with ozone .
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