Chef mapper equipment

The PFGE methodology and analysis were done according to Rivera et al. Reference [26 ]. Briefly, agarose blocks containing leptospiral DNA were prepared and then digested with 30 U of NotI restriction enzyme for 2 hours at 37°C. Salmonella serotype Braenderup H9812 was digested with 50 U XbaI for use as a standard marker. The agarose blocks containing the digested DNA were placed in the wells of the 1% agarose gel (SeaKem Gold) in 0.5X TBE buffer. The run was carried out using the CHEF MAPPER equipment (Bio-Rad Laboratories) for 18 h at 14°C with recirculating 0.5X TBE buffer and under the following conditions: Initial time of 2.16 s, final time of 35.07 s, an angle of 120° and voltage gradient of 6 V/cm. Gels were stained with ethidium bromide (1 ug/mL) for 20 min and documented with Gel-Doc 2000 (Bio-Rad). The images of the gels were analyzed using the GelCompar II program. Dendrograms were created using UPGMA clustering analysis based on band similarity coefficient with optimization of 1.4% and position tolerance of 1.4%. The database with the PFGE profiles of the 65 reference strains of Leptospira spp. was used as a search library for the comparison and identification of serovars of the isolates studied. It was compared with the results obtained by Galloway and Levett for the reference strains [27 (link)].

Bacterial culturing of clinical specimens was performed according to routine operational procedures [5] (Table 1). Bacterial identification was done using the API biochemical identification system (bioMérieux, Marcy-l'Etoile, France) according to the manufacturer's guidelines.
Opportunistic pathogens were considered clinically significant if the organisms grew dominantly on culture media or the same organisms were detected from more than 2 cultures or from different types of samples. And the results were reported when supported by both laboratory data and clinical data.
PFGE was performed with the NotI restriction enzyme (Invitrogen) according to the standard operating procedure for Pulsenet (Pulsenet International) PFGE of Escherichia coli O157: H7, E. coli NON-0157 (STEC), Salmonella serotypes, Shigella sonnei and Shigella flexner. Plugs were submitted to electrophoresis using CHEF Mapper equipment (BioRad). The Lambda PFGE marker (Biolabs) was used in each assay. Images of the pulsetypes were uploaded and analyzed using BioNumerics software, Version 5.1 (Applied Maths).