Plasmids and temporal stability of the extended-spectrum and AmpC beta-lactamase genotype among clinical Salmonella enterica isolates

Objectives: To characterize plasmids implicated in the mobilization of the ESBL and AmpC genes present in clinical Salmonella enterica isolates; as well as to analyze variations in the resistance phenotype in absence of antimicrobial pressure. Methods: Eleven S. enterica isolates carrying bla CTX-M-9 (serovar Virchow, 2 isolates), bla CTX-M-10 (Virchow, 2), bla CTX-M-14 (Enteritidis, 1), bla CTX-M-15 (Gnesta, 1, and S. enterica group C, 1), bla SHV-2 (Livingstone, 1), bla SHV-12 (Enteritidis, 1) and bla CMY-2 (Bredeney, 2) were studied. Characterization of plasmids harbouring ESBL and AmpC genes was carried out by alkaline lysis and S1-PFGE followed by Southern-blot hybridization with specific probes. Genetic surroundings and integron content were investigated. Conjugation and plasmid typing (PBRT) experiments were done. Temporal stability experiments were performed through daily passages (100 days) in the absence of antimicrobials. Weekly tests were done to check the phenotype, the genetic content and the plasmid and beta-lactamase gene variations. Results: IncI1 and IncA/C plasmids carried bla CTX-M-14, bla CTX-M-15 and bla CMY-2 genes, but the bla CTX-M-9 included into an In60 complex integron and the bla CTX-M-10 linked to a phage related element, were found into non-typeable plasmids. The ISEcp1-bla CTX-M-14 -IS903 and ISEcp1-bla CTX-M-15 -orf477 genetic structures were detected. The transference of ESBL/AmpC genes was successful from 8 of the 11 Salmonella isolates into an E. coli recipient strain by conjugation experiments. Other non-beta-lactam resistances were also co-transferred in most of them. In the stability experiments, five of the 11 tested isolates lost the plasmidic ESBL or AmpC genes (bla CTX-M-14 , bla CTX-M-15 , bla SHV-2 , bla SHV-12 and bla CMY-2 ). This fact was associated with the concomitant loss of the complete IncI plasmids that harboured bla SHV-12 or bla CMY-2 in two isolates. Additional non-beta-lactam resistance genes, such as tet(A) and tet(B), and integrons carrying dfrA12 and dfrA16 genes, were lost in two additional isolates. Four of the 11 isolates showed no change after 100 days without antimicrobial pressure. Conclusion: Successful plasmids belonging to different incompatibility groups mobilize ESBL and AmpC encoding genes in S. enterica. The lost of ESBL/AmpC genes in absence of antimicrobial pressure could explain the low prevalence of these beta-lactamases among Salmonella isolates.