NorB, an efflux pump in Staphylococcus aureus strain MW2, contributes to bacterial fitness in abscesses

Abstract

While remaining a major problem in hospitals, Staphylococcus aureus is now spreading in communities. Strain MW2 (USA400 lineage) and other community methicillin-resistant S. aureus strains most commonly cause skin infections with abscess formation. Multidrug resistance (MDR) efflux pumps contribute to antimicrobial resistance but may also contribute to bacterial survival by removal of environmental toxins. In S. aureus, NorA, NorB, NorC, and Tet38 are chromosomally encoded efflux pumps whose overexpression can confer MDR to quinolones and other compounds (Nor pumps) or tetracyclines alone (Tet38), but the natural substrates of these pumps are not known. To determine the role of these efflux pumps in a natural environment in the absence of antibiotics, we used strain MW2 in a mouse subcutaneous abscess model and compared pump gene expression as determined by reverse transcription-PCR in the abscesses and in vitro. norB and tet38 were selectively upregulated in vivo more than 171- and 24-fold, respectively, whereas norA and norC were downregulated. These changes were associated with an increase in expression of mgrA, which encodes a transcriptional regulator known to affect pump gene expression. In competition experiments using equal inocula of a norB or tet38 mutant and parent strain MW2, each mutant exhibited growth defects of about two- to threefold in vivo. In complementation experiments, a single-copy insertion of norB (but not a single-copy insertion of tet38) in the attB site within geh restored the growth fitness of the norB mutant in vivo. Our findings indicate that some MDR pumps, like NorB, can facilitate bacterial survival when they are overexpressed in a staphylococcal abscess and may contribute to the relative resistance of abscesses to antimicrobial therapy, thus linking bacterial fitness and resistance in vivo.

FIG. 1.

Structure of the tet38 and norB loci on the MW2 chromosome. The filled rectangles show the extent of the in-frame deletion and replacement by the cat gene. The horizontal arrows indicate the chromosomal fragments that were cloned in pSK950 to complement the norB and tet38 deletions.

FIG. 2.

Growth curves for MW2 and norB and tet38 mutants in vitro. Cells were grown in BHI broth at 37°C.

FIG. 3.

Competitive growth of norB and tet38 mutants with wild-type strain MW2. The CI is defined as the mutant/wild-type output CFU ratio normalized by using the input CFU ratio. A CI of <1.0 indicates a mutant with a growth disadvantage compared to the wild type. Each open circle indicates a CI for either an abscess (in vivo) or a BHI culture (in vitro). The geometric mean of CIs for each group is indicated by a horizontal bar. Competition experiments were repeated separately at least three times for each group. Competition group A, MW2 norB versus MW2 in vivo; competition group B, MW2 norB versus MW2 in vitro; competition group C, MW2 tet38 versus MW2 in vivo; competition group D, MW2 tet38 versus MW2 in vitro; competition group E, MW2 norB geh::pSK950 versus MW2 geh::pSK950 in vivo; competition group F, MW2 norB geh::pSKnorB versus MW2 geh::pSK950 in vivo.

FIG. 4.

Diagram showing single-copy integration in the geh gene in the chromosome using pSK950. The filled triangle indicates the attB site in geh at which complementing plasmids pSKnorB and pSKtet38 were integrated.