In vivo survival of teicoplanin-resistant Staphylococcus aureus and fitness cost of teicoplanin resistance

Abstract

Glycopeptide resistance, in a set of in vitro step-selected teicoplanin-resistant mutants derived from susceptible Staphylococcus aureus SA113, was associated with slower growth, thickening of the bacterial cell wall, increased N-acetylglucosamine incorporation, and decreased hemolysis. Differential transcriptome analysis showed that as resistance increased, some virulence-associated genes became downregulated. In a mouse tissue cage infection model, an inoculum of 10(4) CFU of strain SA113 rapidly produced a high-bacterial-load infection, which triggered MIP-2 release, leukocyte infiltration, and reduced leukocyte viability. In contrast, with the same inoculum of the isogenic glycopeptide-resistant derivative NM67, CFU initially decreased, resulting in the elimination of the mutant in three out of seven cages. In the four cages in which NM67 survived, it partially regained wild-type characteristics, including thinning of the cell wall, reduced N-acetylglucosamine uptake, and increased hemolysis; however, the survivors also became teicoplanin hypersusceptible. The elimination of the teicoplanin-resistant mutants and selection of teicoplanin-hypersusceptible survivors in the tissue cages indicated that glycopeptide resistance imposes a fitness burden on S. aureus and is selected against in vivo, with restoration of fitness incurring the price of resistance loss.

FIG. 1.

Resistance profiles of teicoplanin-selected mutants. Population analysis profiles of susceptible strain SA113 (•), first-step mutant NM18 (⧫), second-step mutant NM30 (▪), and third-step mutant NM67 (▴).

FIG. 2.

Changes in gene expression upon acquisition and loss of glycopeptide resistance. (A) Transcription of differentially expressed genes in SA113 and the three step-selected teicoplanin-resistant mutants NM18, NM30, and NM67 at early-exponential (OD₆₀₀ = 0.5) and mid-exponential (OD₆₀₀ = 2) phases. (B) Transcription profiles of the same genes from SA113 and NM67 cells recovered from mouse TCF on days 1 and 8 of infection.

FIG. 3.

Bacterial load and host response after infection of tissue cages with strains SA113 and NM67. In vivo bacterial growth and host response over 8 days in tissue cages infected with 10⁴ CFU of S. aureus. Symbols: •, SA113 (n = 5); ▴, NM67 growing in tissue cages (n = 4); ▵, NM67 cleared from tissue cages during infection (n = 3); □, values in uninfected cages. Time zero refers to values before infection. (A) Bacterial load in TCF. (B) MIP-2 concentrations in TCF. (C) Leukocyte numbers in TCF. (D) Viable leukocytes in TCF. Median values are plotted for each group, and the lowest quartiles are indicated. , P < 0.05; , P < 0.01.

FIG. 4.

Teicoplanin resistance profiles after passage in tissue cages. Median values of population analysis profiles on increasing concentrations of teicoplanin for strains SA113 and NM67 isolated from tissue cages on days 1 and 8. Key: filled triangles with solid line, strain NM67 on day 1 (n = 5); filled triangles with dashed line, strain NM67 on day 8 (n = 4); filled circles with solid line, strain SA113 on day 1 (n = 4); filled circles with dashed line, SA113 on day 8 (n = 4).

FIG. 5.

Transmission electron microscopy of SA113 and NM67. (A) Diameter of cytoplasm and cell wall thickness of SA113 and NM67 ex vivo on days 1 and 8 of infection. Bars show the average cytoplasm diameter and cell wall thickness in nanometers. Mean values from at least 20 electron microscopy pictures, made from bacteria that were harvested from three mice infected with either NM67 or SA113, are shown. , P < 0.01 NM67 versus SA113 cell wall thickness on day 1; , P < 0.001 NM67 versus SA113 cytoplasm diameter on day 1; , P < 0.05 NM67 versus SA113 cell wall thickness on day 8. (B) Representative TEM pictures of SA113 (A and B) and NM67 (C and D) on days 1 (left) and 8 (right). Scale bars in the bottom right hand corners of the pictures correspond to 50 nm.

FIG. 6.

Changes in ¹⁴C-labeled N-acetylglucosamine incorporation and hemolysis. (A) ¹⁴C-labeled N-acetylglucosamine incorporation. Key: filled circle with solid line, SA113 on day 1; filled triangle with solid line, NM67 on day 1; filled circle with dashed line, SA113 on day 8; filled triangle with dashed line NM67 on day 8 after infection. Median values from four cages were determined, and the lowest quartiles are indicated for each group. According to analysis of variance for repeated measures, P < 0.05 (,) for NM67 versus SA113 on days 1 and 8, respectively. (B) Hemolysis zones on sheep blood agar. One representative TCF isolate is shown for each strain from days 1 and 8.