E separately inoculated into brain heart infusion broth (Oxoid Ltd., Basingstoke, England) and incubated at 37uC for 4 h. They were then mixed at a ratio of 1:10 (Donor:Recipient by volume) for overnight incubation at 37uC. A 0.1-ml volume of the overnight broth mixture was spread onto a MacConkey agar plate containing sodium azide (100 m/mL) and imipenem (2 mg/ mL).Results Antimicrobial Susceptibility Testing Results for NDM-1 Carrying K. pneumoniae and their TransconjugantsAntimicrobial susceptibility testing results showed that blaNDM-1 carrying K. pneumoniae clinical isolates, 43320 and 44951, from patient 1 and 2 respectively, were resistant to all tested antibiotics (Table 1). Addition of a b-lactamase inhibitor did not increase the susceptibility to b-lactams. The E. coli transconjugants, TCJ-P1 and TCJ-P2, respectively from 43320 and 44951 had a different resistance profile when compared with the corresponding clinical isolates but were still resistant to all b-lactams except aztreonam. Reduced MICs for imipenem and meropenem were observed in both transconjugants. Both transconjugants were susceptible to non-b -lactam antibiotics including ciprofloxacin, gentamicin, tetracycline and trimethoprim/sulfamethoxazole. PFGE of 43320 and 44951 showed that they were unrelated with more than six bands difference. MLST indicated that 43320 and 44951 belonged to ST273 and ST1 respectively (data not shown). The plasmid incompatibility typing initially was positive for a product with a size consistent with the PCR product for IncN. However, subsequent sequencing of the PCR products showed unrelated sequences for a putative IS911 transposase orfA with KpLE2 phage-like element.Molecular Typing for NDM-1 Carrying K. pneumoniae and their TransconjugantsMultilocus sequencing typing (MLST) and Pulsed field gel electrophoresis (PFGE) were performed for both blaNDM-1 carrying K. pneumoniae strains. Sequences of seven housekeeping genes for MLST were obtained according to Diancourt et al., Sequences were compared with those on the MLST web site (http://www. pasteur.fr/recherche/genopole/PF8/mlst/Kpneumoniae.html) developed by Diancourt et al., [15] and alleles and sequence types (STs) were assigned accordingly. If there was a difference in two or more alleles the strains were considered to be unrelated. For PFGE, DNA was prepared as described previously [16]. The restriction enzyme XbaI (New England Biolabs, Beverly, MA, USA) was used at the manufacturer’s suggested temperature. Restriction fragments were separated by PFGE in 1 agarose gel (Bio-Rad, Hercules, CA, USA) in 0.56TBE buffer (45 mM Tris, 45 mM boric acid, 1.0 mM EDTA, pH8.0) for 22 h at 200 V at a MedChemExpress AN-3199 temperature of 14uC, with ramped times of 2 to 40 s using the BioRad CHEF MAPPER apparatus (Bio-Rad Laboratories, Richmond, CA, 1527786 USA). Gels were then stained with ethidium bromide and photographed under ultraviolet light. The resulting genomic DNA profiles, or “order Dimethylenastron fingerprints”, were interpreted according to established guidelines [17]. Plasmid replicon typing was performed for transconjugants [18].Sequence Annotation and Comparison of the Two blaNDM-1 PlasmidsComplete sequencing was performed for the two circular blaNDM-1 plasmids, pTR3 and pTR4, respectively from 43320 and 44951. The results of the assemblies of the two plasmids based on 454 reads were almost identical, in that only seven locations of indels were found between pTR3 and pTR4. Subsequent sequence verifications by Sanger reads have.E separately inoculated into brain heart infusion broth (Oxoid Ltd., Basingstoke, England) and incubated at 37uC for 4 h. They were then mixed at a ratio of 1:10 (Donor:Recipient by volume) for overnight incubation at 37uC. A 0.1-ml volume of the overnight broth mixture was spread onto a MacConkey agar plate containing sodium azide (100 m/mL) and imipenem (2 mg/ mL).Results Antimicrobial Susceptibility Testing Results for NDM-1 Carrying K. pneumoniae and their TransconjugantsAntimicrobial susceptibility testing results showed that blaNDM-1 carrying K. pneumoniae clinical isolates, 43320 and 44951, from patient 1 and 2 respectively, were resistant to all tested antibiotics (Table 1). Addition of a b-lactamase inhibitor did not increase the susceptibility to b-lactams. The E. coli transconjugants, TCJ-P1 and TCJ-P2, respectively from 43320 and 44951 had a different resistance profile when compared with the corresponding clinical isolates but were still resistant to all b-lactams except aztreonam. Reduced MICs for imipenem and meropenem were observed in both transconjugants. Both transconjugants were susceptible to non-b -lactam antibiotics including ciprofloxacin, gentamicin, tetracycline and trimethoprim/sulfamethoxazole. PFGE of 43320 and 44951 showed that they were unrelated with more than six bands difference. MLST indicated that 43320 and 44951 belonged to ST273 and ST1 respectively (data not shown). The plasmid incompatibility typing initially was positive for a product with a size consistent with the PCR product for IncN. However, subsequent sequencing of the PCR products showed unrelated sequences for a putative IS911 transposase orfA with KpLE2 phage-like element.Molecular Typing for NDM-1 Carrying K. pneumoniae and their TransconjugantsMultilocus sequencing typing (MLST) and Pulsed field gel electrophoresis (PFGE) were performed for both blaNDM-1 carrying K. pneumoniae strains. Sequences of seven housekeeping genes for MLST were obtained according to Diancourt et al., Sequences were compared with those on the MLST web site (http://www. pasteur.fr/recherche/genopole/PF8/mlst/Kpneumoniae.html) developed by Diancourt et al., [15] and alleles and sequence types (STs) were assigned accordingly. If there was a difference in two or more alleles the strains were considered to be unrelated. For PFGE, DNA was prepared as described previously [16]. The restriction enzyme XbaI (New England Biolabs, Beverly, MA, USA) was used at the manufacturer’s suggested temperature. Restriction fragments were separated by PFGE in 1 agarose gel (Bio-Rad, Hercules, CA, USA) in 0.56TBE buffer (45 mM Tris, 45 mM boric acid, 1.0 mM EDTA, pH8.0) for 22 h at 200 V at a temperature of 14uC, with ramped times of 2 to 40 s using the BioRad CHEF MAPPER apparatus (Bio-Rad Laboratories, Richmond, CA, 1527786 USA). Gels were then stained with ethidium bromide and photographed under ultraviolet light. The resulting genomic DNA profiles, or “fingerprints”, were interpreted according to established guidelines [17]. Plasmid replicon typing was performed for transconjugants [18].Sequence Annotation and Comparison of the Two blaNDM-1 PlasmidsComplete sequencing was performed for the two circular blaNDM-1 plasmids, pTR3 and pTR4, respectively from 43320 and 44951. The results of the assemblies of the two plasmids based on 454 reads were almost identical, in that only seven locations of indels were found between pTR3 and pTR4. Subsequent sequence verifications by Sanger reads have.
