Pulsed-field gel electrophoresis (PFGE) was performed with the XbaI restriction enzyme (New England Biolabs, Beverly, MA, USA) as described previously.34 (link) PFGE profiles were analyzed with InfoQuest FP software version 4.5 (Bio-Rad Laboratories Inc., Hercules, CA, USA). In order to establish the epidemiological relationship among the isolates from the electrophoretic patterns, the Dice coefficient was used and clustering was based on the unweighted pair group method with arithmetic mean with a 1% tolerance in band position differences. The isolates were considered to belong to the same epidemiological group when the PFGE-XbaI profiles showed ≥80% of homology, adapting the criteria described by Tenover et al.35 (link) Multi-locus sequence typing (MLST) was carried out by amplification and sequencing of the 7 E. coli housekeeping genes as described previously.36 (link) The database available at http://mlst.warwick.ac.uk/mlst/dbs/Ecoli/ was used for assigning sequence types (STs) and clonal complexes (CCs). Classification of isolates into E. coli phylogenetic groups was done using a previously described triplex PCR-based protocol.37 (link)
Protocol detail
Genomic Fingerprinting of E. coli Isolates
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Based pair
Clonal
E coli
Electrophoretic
Housekeeping genes
Pulsed field gel electrophoresis
Restriction enzyme
Tolerance
Triplex pcr
Corresponding Organization :
Other organizations : Universitat de Barcelona, Barcelona Institute for Global Health, Manhiça Health Research Centre, Instituto Nacional de Saúde
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Protocol cited in 2 other protocols
Variable analysis
independent variables
- Restriction enzyme (XbaI)
- PFGE profiles
- Epidemiological relationship among isolates
- Sequence types (STs) and clonal complexes (CCs)
- Phylogenetic groups of E. coli isolates
- Experimental protocol described previously (Ref. 34)
- Clustering method (Dice coefficient, unweighted pair group method with arithmetic mean)
- Homology criteria (≥80%) for epidemiological grouping (Ref. 35)
- MLST protocol for housekeeping genes (Ref. 36)
- E. coli MLST database for assigning STs and CCs
- Triplex PCR-based protocol for phylogenetic grouping (Ref. 37)
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