Isolates resistant to www.selleckchem.com/products/azd2014.html tetracycline and at least three additional antibiotics, but sensitive to gentamicin (which is needed to kill extracellular bacteria in the invasion assays), were then screened for the presence of the Salmonella genomic island 1 (SGI-1) and tetracycline resistance genes known to occur in Salmonella (tetA, B, C, D, and G). The SGI-1 is a 43
kb stable chromosomal integron often found in DT104, and it encodes several antibiotic resistance genes as well as hypothetical genes that have a potential association with virulence [16–18]. The SGI-1 was identified in all DT104 isolates but in none of the DT193 isolates. All the DT104 isolates encoded a single tetracycline resistance gene, tetG, while VX-809 research buy the DT193 isolates encoded the following combinations: tetA; tetA, B, C, and D; or tetB, C, and D. Representatives of each tet-resistance gene combination were selected at random for further study click here (Table 1). Table 1 Characterization of antibiotic resistance profiles and tetracycline resistance genes in eight S. typhimurium isolates Isolate Phagetype Resistance profile tet gene(s) amp chlor gent kan strp tet tetA tetB tetC tetD tetG 1434 DT193 + + – + + + + – - – - 5317 DT193 + + – + + + + – - – - 752 DT193 + + – - + + + – - – - 1306 DT193 + + – + + + + + + + – 4584 DT193 + + – + + + – + + + – 530 DT104
+ + – - + + – - – - + 290 DT104 + + – + + + – - – - + 360 DT104 + + – - + + – - – - + Selection of antibiotic concentrations Growth curves were determined for each of the eight isolates over a range of tetracycline concentrations (0–256 μg/ml). The growth curve for isolate 1434, which is representative of all the isolates, is shown OSBPL9 in Figure 1. Tetracycline concentrations between 1–128 μg/ml did not prevent
growth, and this range was considered sub-inhibitory for this study. No significant change in growth due to antibiotic addition was observed between 1–32 μg/ml of tetracycline. Subsequent invasion and gene expression analyses were performed using several concentrations of tetracycline within this range (0, 1, 4, and 16 μg/ml) in order to assess if an effect on invasion was concentration dependent. Figure 1 Representative growth curve of multidrug-resistant S . Typhimurium exposed to various concentrations of tetracycline. Serial two-fold dilutions of tetracycline (0–256 μg/ml) were added at OD600 = 0.15 to each of the eight isolates to determine the effect of tetracycline exposure on growth. The growth curve of isolate 1434 is shown. Tetracycline induces invasion in a subset of isolates during early-log phase Regulation of the invasion process is initiated during early-log phase of growth , and Salmonella becomes fully invasive during the late-log phase . Cellular invasion assays were performed using isolates grown to early-log phase (OD600 = 0.