Involvement Of Two Efflux Pumps In Tigecycline Resistant S. aureus Mutants

HERRERA, M.; DI GREGORIO, S.; FERNANDEZ, S.; POSSE, G.; MOLLERACH, M.; DI CONZA, J.

Boston

Congreso; ASM Microbe 2016 - ICAAC 2016; 2016

American Society for Microbiology

Background: Staphylococcus aureus has an extraordinary ability to develop antimicrobial resistance. Previously, we reported the in vitro selection of tigecycline resistant mutants (TRM) which showed an increase in tigecycline (TIG) MICs of 128-fold. The efflux phenotype was predicted by ethidium bromide MIC determination (64-128 μg/ml). Additionally, we demonstrated that MIC for both compounds showed a decrease greater or equal than 4 dilutions in the presence of 20 μg /ml reserpine (an efflux pump inhibitor). The aim of this study was to analyze the involvement of two efflux pumps implied in glycylcicline and tetracyclines extrusion (mepA and norB) in two TRM strains analyzed previously (94159m and 2028m). Methods: Efflux pump genes were amplified by PCR and sequencing. Reverse transcription-Quantitative real-time-PCR (RT-qPCR) was performed to evaluate the expression of mepA and norB genes. Data was expressed as normalized relative quantities (NRQ) using gyrB and pta as reference genes. All experiments were performed by triplicate. Statistical analysis of the data was performed using Student t test (alfa=0.05).Results: The nucleotide sequence of mepA gene (1700 bp) in the parental strains 94159p and 2028p showed 100 % of identity with the corresponding reference sequences (S. aureus Accession number HE579073.1 and CPO12119.1). By contrast, for both mutant strains (94159m and 2028m) a transition (C to T) was observed in the position 211 of the sequence, displaying the Thr29Ile mutation in the translated amino acid sequence. In addition 2028m harbored the transition A to G in the position 985 generating the Glu287Gly mutation.NRQ values of mepA gene were significantly higher in both mutant strains (94159m and 2028m) when compared to parental strains (p<0.0001 and p=0.0009 respectively). On the other hand, NRQ values of norB gene were significantly decreased in mutant strain 2028m when compared to parental strain 2028p (p=0.0007), and no difference was detected for the pair 94159p-94159m (p=0.1093).Conclusions: This study demonstrates the involvement of efflux pumps in TRM. We identified a derived, charge-changing amino acid mutation in MepA and an increase in mepA gene expression which would be related to the development of TIG resistance in these mutants. The decrease in norB expression observed might alter the extrusion of other compounds.