Computational protocol: Molecular epidemiology of carbapenem resistant gram-negative bacilli from infected pediatric population in tertiary - care hospitals in Medellín, Colombia: an increasing problem

Similar protocols

Protocol publication

[…] The presence of carbapenemases was evaluated through PCR amplification of genes blaKPC, blaVIM, blaIMP, blaNDM and blaOXA-48, using previously described primers and conditions [, ]. After PCR amplification, forward and reverse sequencing was performed. Sequences were compared with those available at GenBank (www.ncbi.nlm.nih.gov/blast/) and Lahey database (http://www.lahey.org/Studies/).Pulse-field gel electrophoresis (PFGE) was performed using 50 U of SpeI, 20 U of XbaI and 50 U XbaI restriction enzime (Thermo Scientific, United States) for P. aeruginosa, K. pneumoniae and E. cloacae, respectively. DNA fragment patterns were normalized using the bacteriophage Lambda ladder PFGE marker (New England Biolabs, UK). Electrophoresis was performed on a CHEF DR III (Bio-Rad Laboratories, Hercules, CA) at 11 °C, angle 120° and voltage gradient 6 V/cm. Cluster analysis was performed using the Dice coefficient in BioNumerics software version 6.0 (Applied Maths, Sint-Martens-Latem, Belgium). Dendrograms were generated by the unweighted pair group method using average linkages (UPGMA), with 1 % tolerance and 0.5 % optimization settings. A similarity cutoff of ≥80 % was used to define genetically related strains.Multilocus sequence typing (MLST) was performed using the methodology previously described on a subset of isolates representing the most frequent PFGE patterns in P. aeruginosa and K. pneumoniae [, ]. Allele numbers and sequence types (ST) were assigned using the database maintained at http://pubmlst.org/paeruginosa/ and http://bigsdb.web.pasteur.fr/klebsiella/klebsiella.html. […]

Pipeline specifications

Software tools BioNumerics, BIGSdb
Databases PubMLST
Application De novo sequencing analysis
Organisms Homo sapiens, Enterobacter cloacae, Pseudomonas aeruginosa, Klebsiella pneumoniae
Diseases Bacterial Infections, Infection, Pneumonia