Mic testing was performed in duplicate where mic was

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strains. MIC testing was performed in duplicate, where MIC was defined as the drug concentration showing no visible growth. In order to evaluate resistant strains for antibiotic inactivation, cultures containing 5 ml of 50% TSB in the presence and absence of 20 m g/ml of antibiotic were inoculated with 3–5 single isolated colonies of strain of interest and cultures were grown at 30 u C for 5 days. Uninoculated controls were also prepared. After growth at 30 u C for 5 days, cultures were centrifuged for 20 minutes at 16,800 6 g and conditioned media was collected. One of the following susceptible organisms/test organisms was used for antimicrobial susceptibility testing (antimicrobial disk diffusion assay): Bacillus subtilis , Micrococcus luteus and Staphylococcus saprophy- ticus ATCC 15305. Inocula of test organisms were prepared to the 0.5 McFarland standard using the direct colony suspension method according to the Clinical and Laboratory Standard Institute guidelines [51]. Susceptible organisms were plated on LB Agar and conditioned media (20–30 m l) was spotted on sterile paper disks prior to incubation at 37 u C overnight. Inactivation was defined as complete absence of a zone of inhibition. B. paraconglomeratum LC44 Genome Sequencing and Assembly B. paraconglomeratum LC44 was grown in 5 ml 50% TSB at 30 u C for 5 days. Genomic DNA was isolated using QIAGEN DNeasy Blood and Tissue Kit 250 (Qiagen, Germany) with one modification of the manufacturer’s protocol for cell lysis: 4 m l of RNase (100 mg/ml in Buffer TE) was added to the reaction mixture and incubated at room temperature for 2 minutes before washing the spin column. Genomic DNA was submitted for shotgun sequencing to Roche 454 Life Sciences Genome Sequencer at Farncombe Metagenomics Facility, McMaster University. Approximately one quarter of a 454 PTP was used for Titanium pyrosequencing on a 454 GS FLX. Additionally, 8059104 Illumina GAIIx 71 bp paired end reads were sequenced by Ambry Genetics (Aliso Viejo, California). After aggressive quality trimming and filtering, the approximately 5.5 million remaining Illumina reads (about 70 6 coverage) and 262995 454 reads (about 23 6 coverage) were assembled using MIRA version 3.4 with the ‘-job = denovo,genome,454,accurate,solexa’ switches [52]. The resulting contig sequences of the assembly were deposited in Genbank as a WGS project under accession AGSO00000000. The protein sequence of E. coli MPH (2 9 )-Ia (accession BAA03776) was used to query the assembled sequences using translated blast in order to find a gene responsible for the observed macrolide phosphotransferase activity. A good candidate was found on contig AGSO00000004 beginning at bp 76533 and a blastx search was performed against the NCBI non-redundant protein database where the top hits corresponded to sequences annotated as putative APH and MPH proteins. Antibiotic Resistance in Cave Bacteria PLoS ONE | 9 April 2012 | Volume 7 | Issue 4 | e34953
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Cloning, Expression and Purification of Macrolide Phosphotransferases The candidate macrolide phosphotransferase from B. paracon- glomeratum ( mphE ) was synthesized by GenScript (USA) with codon optimization for E. coli
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