PMC:1069006 / 4483-8706 JSONTXT

{"project":"2_test","denotations":[{"id":"15781495-11997336-76989425","span":{"begin":217,"end":219},"obj":"11997336"},{"id":"15781495-9521922-76989426","span":{"begin":525,"end":527},"obj":"9521922"},{"id":"15781495-9521921-76989426","span":{"begin":525,"end":527},"obj":"9521921"},{"id":"15781495-9521923-76989426","span":{"begin":525,"end":527},"obj":"9521923"},{"id":"15781495-10556321-76989427","span":{"begin":1003,"end":1005},"obj":"10556321"},{"id":"15781495-11677609-76989428","span":{"begin":1087,"end":1088},"obj":"11677609"},{"id":"15781495-11677608-76989428","span":{"begin":1087,"end":1088},"obj":"11677608"},{"id":"15781495-12644504-76989428","span":{"begin":1087,"end":1088},"obj":"12644504"},{"id":"15781495-9254694-76989429","span":{"begin":1506,"end":1508},"obj":"9254694"},{"id":"15781495-12520011-76989430","span":{"begin":1740,"end":1742},"obj":"12520011"},{"id":"15781495-3306260-76989431","span":{"begin":2020,"end":2021},"obj":"3306260"},{"id":"15781495-10665125-76989432","span":{"begin":2069,"end":2070},"obj":"10665125"},{"id":"15781495-11435108-76989433","span":{"begin":2191,"end":2193},"obj":"11435108"},{"id":"15781495-11677609-76989434","span":{"begin":2332,"end":2333},"obj":"11677609"},{"id":"15781495-11677608-76989435","span":{"begin":2334,"end":2336},"obj":"11677608"},{"id":"15781495-12523625-76989436","span":{"begin":3833,"end":3835},"obj":"12523625"},{"id":"15781495-12523625-76989437","span":{"begin":4106,"end":4108},"obj":"12523625"}],"text":"MATERIALS AND METHODS\n\nGenome sequencing and analysis\nS.Choleraesuis strain SC-B67 was isolated from a patient with sepsis in 2002. Whole-genome libraries in pUC18 were prepared from genomic DNA as described earlier (12). Random clones were sequenced by using dye-terminator chemistry and data collected on MegaBASE 1000 automated sequencers. The total number of reads (\u003e200 bp) was 66 494, with an average read length of 540 bp. The Phred/Phrap/Consed software package was used for quality assessment and sequence assembly (13–15). Gaps were filled by sequencing opposite ends of linking clones and the use of several PCR-based techniques and primer walking methods. The final assembly was verified by comparison with restriction-enzyme digest patterns of I-CeuI, AvrII and XbaI. Furthermore, we performed PCR on genomic DNA to amplify regions covering the pseudogenes. All the sequence alterations were confirmed by re-sequencing the PCR products.\nThe potential CDS were established with GLIMMER 2.1 (16) and the annotated open reading frames (ORFs) of three known Salmonella genomes (9–11); both predicted CDS and putative intergenic sequences were subjected to further manual inspections. ORFs that contain insertion, deletion or mutation to a stop codon compared with those known Salmonella genes were selected as pseudogenes. To the 4445 determined ORFs, exhaustive BLAST searches with an incremental stringency against NCBI non-redundant protein database were performed to determine their homology (17). To further investigate the function of each protein we used the InterProScan to search against the InterPro protein family database, which included PROSITE, PRINTS, Pfam, ProDom, SMART, TIGRFAMs, PIR SuperFamily and SUPERFAMILY (18). The up-to-date KEGG pathway database () was used for pathway analysis. Genes from 127 bacteria were gathered and classified into 127 pathways, to which we searched the pseudogenes in the BLAST suite, and the best hit was selected. The BLAST search criteria were as follows: (1) ∼30% of the subject sequence was aligned and (2) E-value = 10−10 or less. Putative aliens were detected by finding anomalous gene regions in genomic characterizations (19), and the pathogenic islands were further determined according to their function annotation and comparison with other Salmonella genomes (9,10). Unique regions of a genome were identified as sequences that have no counterpart in another compared genome with MegaBlast search. Genome used for comparison are from GenBank and their accession numbers are: NC_003143, NC_004061, NC_002528, NC_004741, NC_004631, NC_004545, NC_000913, NC_004431, NC_004088, NC_003198, NC_002695, NC_002655, NC_004344, NC_004337 and NC_003197.\n\nElectron microscopy\nScanning electron microscopy was performed to examine the flagella of S.Choleraesuis SC-B67 and S.Typhimurium LT2. In preparation, bacteria were collected from the culture plate and suspended directly to 3% glutaraldehyde–2% paraformaldehyde mixture in 0.1 mol/l cacodylate buffer (pH = 7.4) for fixation at room temperature for 1 h. After adhering to poly-l-lysine coated glass coverslips, they were then post-fixed in 1% osmium tetroxide in the same buffer at 4°C for 1 h. After dehydration with graded ethanol, the samples were critical point dried, mounted onto the specimen stub, coated with Pt/Pd alloy, and examined with Hitachi S-5000 electron microscope at 3 kV.\n\nInhibition assay and western blot analysis\nMICs were determined by the standard agar dilution method on the Mueller-Hinton medium. The MIC was defined as the lowest concentration of the drug that completely inhibited visible growth after inoculation for 18 h at 37°C. MICs of ciprofloxacin were also determined in the presence of the efflux pump inhibitor, Phe-Arg-β-naphthylamide (Sigma), at the concentration of 80 μg/ml (22). The efflux pump protein, AcrA, expression in Salmonella strains was analyzed by western blot hybridization. Fluoroquinolone-susceptible S.Typhimurium BN18 and in vitro-selected resistant mutants BN18/21, BN18/41 and BN18/71 were used as controls in these experiments (22). For comparison, a fluoroquinolone-susceptible S.Choleraesuis isolate SC-B42, was also examined by these methods."}