Methods Isolates The 1327 non-duplicate isolates were obtained sequentially from 13 healthcare facilities in Kenya between 1992 and 2011 (19-year period) from 654 hospitalized and 673 non-hospitalized patients. These isolates comprised of 451 strains from patients with urethral tract infections (UTI) and those with urinary catheters while 371 were from blood of patients with septicemia. Another 505 strains were from fecal specimens of patients with loose stool, watery and bloody diarrhea. Only one isolate per specimen per patient was included for further analysis.
Among the isolates investigated in this study, MK0683 order 912 had been analyzed for bla genes in a a past study  while 27 had been analyzed for selected genetic elements . Ethical clearance to carry out this study was obtained from the KEMRI/National Ethics Committee (approval number SSC No. 1177). Antimicrobial susceptibility profiles Susceptibility profiles for all
isolates were determined using antibiotic discs (Cypress diagnostics, Langdorp, Belgium) on Mueller Hinton agar (Oxoid, Louis, Mo. USA) using the Laboratory Standards Institute guidelines (CLSI) . Detection of genetic elements Figure 1 illustrates the strategy used for detection and characterization of integrons and transposons. Detection of class 1, 2 and 3 and determination of carriage of 3’-conserved sequences (3’-CS) in class 1 integrons was done as described before [34, 35]. Class 1 integron variable cassette region (VCR), the region Selleckchem BVD-523 in which the resistance gene cassettes are integrated, was amplified as previously described by Dalsgaard et al. while that of class 2 integrons was amplified as described Telomerase by White et al.. The VCRs of integrons lacking the typical 3’-CS was determined using a PCR walking strategy published before . Identification of integron cassette identity was done using a combination of restriction fragment length polymorphism (RFLP), sequencing and published bioinformatics tools [38, 39].
Detection of the ISEcp1, ISCR1, Tn21 and Tn7 elements was done as described in published studies [34, 35]. Analysis for Tn21 transposition genes:- tnpA, tnpR and tnpM genes was done as previously described by Pearson et al.. The primers used in this study are presented in Table 10. Table 10 Primers for screening for genetic elements and resistance genes and for analysis for physical linkages among such elements and selected resistance genes Target Gene/region Primer name 5′-3′ sequence Annealing Temperature Expected product size (bp) Gene accession Number Integrons intI1 INT-1 F GTTCGGTCAAGGTTCTG 50 923 U12338 INT-1R GCCAACTTTCAGCACATG intI2 INT-2 F ATGTCTAACAGTCCATTTT 50 450 AJ001816.