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In June 2008, we found nine A. baumannii isolates that were susceptible to AN by VITEK 2 but that showed a double zone of inhibition to AN by a disk diffusion test. AN MIC was determined by the agar dilution method according to the NCCLS guideline [ 17 ] and showed an AN MIC of >512 μg/ml. Because of its extraordinarily high AN MIC, we investigated 16S rRNA methylase genes ( rmtA, rmtB, rmtC , and armA ) by PCR as described previously [ Womens 41510718 Velvet Boots Buffalo Outlet With Paypal Order oPOkyp
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]. The nine isolates were shown to harbor armA . Pulsed-field gel electrophoresis (PFGE) analysis was carried out to investigate clonality, because they were isolated from the neurosurgery ward and the pediatric intensive care unit (ICU). Briefly, the whole-cell DNA of the A. baumannii isolate was digested with the Apa I restriction enzyme for 18 hr at 25°C. Electrophoresis was carried out with a CHEF-DR III Variable Angle System (Bio-Rad) through a 1% agarose gel in 0.5X Tris/borate/EDTA buffer at 14°C, a voltage of 6 V/cm, and a switch angle of 60°, using pulse times ranging from 2 to 20 sec for 22 hr. Lambda Ladder PFGE Marker (New England Biolabs) was used as a DNA molecular weight marker [ 18 ]. The PFGE profiles of all but one of the isolates revealed a closely related pattern (Fig. 1 Womens Twiggy Ankle Jeans James Jeans Clearance Wholesale Price uvVCW29
).

In order to investigate the mechanism underlying this phenomenon, we performed several experiments:

First, to check for purity, we selected a single colony from the A. baumannii and E. coli transconjugant in Mueller–Hinton broth, and repeated the disk diffusion test. Both showed the same phenomenon with a single colony.

Second, to investigate if this phenomenon can occur in other species, we tested the AN susceptibility of the Serratia marcescens isolate harboring armA and its transconjugant ( E. coli J53 harboring armA ) using VITEK 2, a disk diffusion test, and the agar dilution method. Conjugation experiment was performed with an azide-resistant E. coli J53 as a recipient strain, by filter mating. Transconjugants were selected on LB agar plates supplemented with sodium azide (150 μg/ml) and amikacin (32 μg/ml). From the transconjugant, DNA was extracted and the transfer of armA was confirmed by PCR. By VITEK 2, the S. marcescens was determined to be resistant to AN, whereas the transconjugant harboring armA was susceptible to AN. Upon testing with a disk diffusion test, the S. marcescens showed a zone of complete resistance, while the transconjugant revealed a double zone of inhibition as shown by A. baumannii . However, the AN MICs of the two isolates were both high (>512 μg/ml) (Table 1 ).

Third, to investigate whether this phenomenon was associated with induction, we selected resistant subpopulations (colonies grown around the AN disk) and repeated the AN susceptibility and disk diffusion tests. Again, they showed a double zone of inhibition, suggesting that induction was not the cause.

To evaluate the genetic relatedness between W1481 and the other Fusobacterium genomes the average nucleotide identity (ANI) ( Konstantinidis and Tiedje 2005 ) was calculated based on the method proposed by Goris et al. (2007) . Two-way BLAST was chosen and only forward and reversed-matched orthologs were used in the calculations. For the robustness, the BLAST match have been set at least 50% identity at the nucleotide and amino acid level and a sequence coverage of at least 70%.

The calculation of average amino acid identity (AAI) was performed by the method as described by Konstantinidis and Tiedje (2005) . The RAST annotated protein-coding sequences of the W1481 genome was used as the reference for comparison against other Fusobacterium genomes (also annotated using RAST), using the BLAST search to determine the conserved genes. The cut-off was set at ≥30% sequence identity and ≥70% sequence coverage at the amino acid level for the BLAST search. The average of the amino acid identity of all conserved genes between a pair of genomes was computed to measure the genetic relatedness between them.

All genomes sequences were submitted to IslandViewer ( Langille and Brinkman 2009 ) for genomic island (GI) prediction. IslandViewer implements a sequence composition based approaches derived from other validated GI prediction software packages, which were SIGI-HMM ( Waack et al. 2006 ) and IslandPath-DIMOB ( Hsiao et al. 2003 ). Both the composition based approach were shown to have a higher than 86% overall accuracy compared with other prediction software. IslandViewer also integrated IslandPick, which was developed based on comparative genomics approach ( Langille et al. 2008 ). To cluster these GIs for comparison across different F. nucleatum strains, the GI nucleotide sequences were clustered using BLASTClust ( Altschul et al. 1990 ) with the thresholds of at least 50% sequence identity and 50% sequence coverage.

Homology searches were performed on the protein sequences of 36 Fusobacterium strains against the Virulence Factors Database (VFDB) ( Chen et al. 2005 , 2012 ), by applying BLASTP of the BLAST software package ( Altschul et al. 1997 ). The BLAST hits were processed using in-house Python script filtering and accepting only orthologs at the threshold of at least 50% sequence identity and 50% sequence completeness between the query and subject sequences. Instead of using the tabular comparison for pathogenomic composition provided in VFDB release of 2012, the data collected from VFDB was used to construct a graphical heat map representation using our in-house R Scripts, allowing for the comparison of the virulence gene profiles across all Fusobacterium strains.

Functional ortholog clustering was performed using PGAP (Pan-genome Analysis Pipeline) ( Zhao et al. 2012 ). The protein sequences of 21 F. nucleatum strains were used as the input file. The orthologs among these 21 strains were searched using the Gene Family (GF) method included in the PGAP pipeline. Protein sequences of each strain were clustered together and denoted with strain identifiers. BLASTALL program ( Altschul et al. 1990 ) was performed among the protein sequences with the minimum score value of 50 and E-value at 1 e-8 . The filtered BLAST results were clustered by MCL algorithm ( Enright et al. 2002 ). In order to group the same genes into the same cluster, the global match region must have at least 70% of the longer gene protein sequence (coverage) and 40% sequence identity.

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