Each isolate demonstrated variable degrees of antibiotic resistance gene silencing . Pair-wise growth competition assays were performed between silent isolates and the wild-type isolates expressing all antibiotic resistance genes. Isolate L5 had a slight in vitro cost of -2.1% ± 1.7% per generation whilst isolates L4 and L7 had slight fitness advantages of +1.1 ± 1.4% and +1.2% ± 0.5% per generation, respectively. However, the statistical significance of these find more results was low and overall the impact of silencing of pVE46
genes on fitness appeared negligible. The in vivo ability of isolate L5 to colonize the pig gut was found to be comparable to that of 345-2RifC(pVE46) (Figure 2). Figure 2 learn more Recovery of E. coli 345-2RifC(pVE46) (squares), E. coli L5 (diamonds), E. coli 345-2RifC(RP1) (triangles) and E. coli P2 (circles) from pig faeces following oral inoculation of six animals. There was statistically no difference in recovery levels between 345-2RifC(pVE46) and L5 (ANOVA 0.5628, p = 0.4546). However, P2 was recovered significantly more frequently than 345-2RifC(RP1) (ANOVA 15.3169, p = 0.0002). Table 3 Characteristics of bacterial strains and plasmids
used in this study Plasmids Resistance Profile1 Resistance Genotype Inc Group Reference or source pVE46 AMP, STR, BI 10773 solubility dmso SUL, TET bla OXA-2, sul1, aadA1, tet(A) N  R46 AMP, STR, SUL, TET bla OXA-2 × 2, sul1, aadA1, tet(A) N  RP1 AMP, KAN, TET bla TEM-2, aphA, tet(A) P  PUB307
KAN, TET aphA, tet(A) P  N3 STR, SUL, TET sul1, aadA1, tet(A) N  Bacterial Strains Phylogenetic Group 345-2RifC RIF RpoB H526Y B1  343-9 NA D  99-24 NA D  99-40 NA B2  K12 JM109 NAL NA A Promega, Southampton, UK L52 RIF bla OXA-2, sul1, aadA1, tet(A) B1  L42 RIF, TET bla OXA-2, sul1, aadA1, tet(A) B1  L72 AMP, RIF, SUL bla OXA-2, sul1, aadA1 B1  P13 KAN, RIF bla TEM-2 B1  P23 RIF bla TEM-2, aphA, tet(A) B1  1AMP, ampicillin; KAN, kanamycin; NAL, nalidixic acid; RIF, rifampicin; STR, streptomycin; SUL, sulfamethoxazole; TET, tetracycline; NA, not applicable 2345-2RifC strain with pVE46 encoding silent antimicrobial Galactosylceramidase resistance genes 3345-2RifC strain with RP1 encoding silent antimicrobial resistance genes In contrast, antibiotic resistance gene silencing had a significant effect on the fitness of E. coli 345-2RifC(RP1). The silent isolates P1 and P2 (Table 3) both had fitness advantages of +2.5 ± 0.5% and +4.1 ± 3.7% in vitro, respectively. P2 was also able to colonize the pig gut better than 345-2RifC(RP1) (Figure 2). Surprisingly, antibiotic resistance gene silencing did not confer a fitness advantage on isolates carrying the pVE46 plasmid, in vivo or in vitro.