Of the 96 isolates, a number of strains overlapped in terms of motility phenotype: 24 had flagellar and twitching motility, 27 had only twitching motility, 47 had only swarming motility and a total of 45 were non motile. Given the complex
phenotypic diversity of the clinical isolates based on direct observations we recognized the need for a rational approach to selecting the most appropriate isolates for further study. We adopted RAPD as a convenient and quick genotyping method that allowed us to characterise the heterogeneity in the group, using a cut off value of 85% similarity as a threshold to compare strains. Primer 10514 generated a total of 22 different profiles (Table 3), fifteen of which contained more than one isolate. Regorafenib Primer 10514-generated profiles were cross- referenced with those of primer 14306 and showed BI 10773 mw that similar profiles were generated with both primers. We noted variations in surface attachment ability and in motility among strains and we selected strains based upon both genotypic and phenotypic characteristics, i.e. strains that represented
similar RAPD groupings and also based upon the degree of biofilm production. selleck compound Twenty genotypically distinct isolates were thus selected for further study (Table 4, column1). Table 4 Correlation of the swimming phenotype of 20 selected clinical Pseudomonas aeruginosa isolates with the presence of fliC
gene and correlation of the twitching phenotype with the presence of the pilA gene Fenbendazole group. Isolate Swimming motility fliC gene Twitching motility pilA gene group 1 + + + II 3 + + + II 7 – + – I 17 + + + I 26 + + + I 29 – + – I 30 – + – I 33 + + – I 38 + + + I 40 + + + I 41 + + – - 46 – + – I 48 – + – I 54 + + – - 55 + + – - 64 + + + II 72 – + – V 80 – + – I 85 – + – I 94 – + – I P. aeruginosa CF isolates exhibit a lack of correlation between motility phenotype and genotype The observed phenotypic differences in twitching motility led us to consider whether non-twitching isolates were inherently non-motile or whether they possessed the capability to be motile but did not express it. Pilin alleles and associated gene(s) are located in a common chromosomal locus between the conserved pilB and tRNA Thr genes [18]. The presence of various tfp accessory genes located upstream of pilA determines amplicon size, thus allowing the delineation of five TFP groups [18, 31]. Seven twitching efficient and 13 twitching deficient isolates were selected (Table 4) and we determined whether or not pilA, the type IV pilus (TFP) gene responsible for the PilA structural protein, was present in the isolates. Thirteen isolates yielded ~2.8 kbp amplicons with the pilB and tRNAThr primers [31], thus the majority of the CF isolates fell into TFP group I (tfpO). Amplicons of ~1.