Staphylococcus aureus phenotype switching: an effective bacterial strategy to escape host immune response and establish a chronic infection

Abstract

Staphylococcus aureus is a frequent cause for serious, chronic and therapy-refractive infections in spite of susceptibility to antibiotics in vitro. In chronic infections, altered bacterial phenotypes, such as small colony variants (SCVs), have been found. Yet, it is largely unclear whether the ability to interconvert from the wild-type to the SCV phenotype is only a rare clinical and/or just laboratory phenomenon or is essential to sustain an infection. Here, we performed different long-term in vitro and in vivo infection models with S. aureus and we show that viable bacteria can persist within host cells and/or tissues for several weeks. Persistence induced bacterial phenotypic diversity, including SCV phenotypes, accompanied by changes in virulence factor expression and auxotrophism. However, the recovered SCV phenotypes were highly dynamic and rapidly reverted to the fully virulent wild-type form when leaving the intracellular location and infecting new cells. Our findings demonstrate that bacterial phenotype switching is an integral part of the infection process that enables the bacteria to hide inside host cells, which can be a reservoir for chronic and therapy-refractive infections.

Figure 1. Staphylococci survive within cultured host cells for 28 days and change phenotypes and virulence factor expression

1. Epithelial cells (A549) were infected with different S. aureus strains (6850 or 628) and analysed for 28 days. The number of viable intracellular persisting bacteria was determined weekly by lysing host cells, plating the lysates on agar plates and counting the colonies that have grown on the following day (n = 3, ±SEM). Electron micrographs of infected cells were performed directly after and 28 days post-infection showing morphological intact staphylococci within epithelial cells.

2. Percentage of small and very small (SCV) phenotypes (<5 and <10-fold smaller than those of the wild-type phenotype, respectively) recovered during the time-course of 28 days (n = 3, between 200 and 500 colonies examined in each sample, ±SEM). Photographs of recovered colonies were performed directly after and 28 days post-infection showing appearance of small and SCV colonies.

3. Changes in bacterial gene expression (strain 6850) for fibronectin binding protein A (fnbA), α-haemolysin (hla) and agr during the course of infection were determined by real-time PCR (n = 5, ±SD).

4. Changes in host cell response measured by the expression of CCL5, CXCL11 and ICAM-1 in the time course after infection with strain 6850 (n = 6). *p ≤ 0.05, **p ≤ 0.01 and ***p ≤ 0.001 in comparison with values from uninfected cells.

Figure 2. Staphylococci survive within different types of primary isolated human host cells

1. Primary isolated human cells (HUVECs, osteoblasts and macrophages) were infected with the wild-type S. aureus strain 6850 and analysed for seven consecutive days. The number of viable intracellular persisting bacteria was determined daily and was dramatically reduced in HUVECs and osteoblasts, but for both cell types, we found living intracellular bacteria through 7 days post-infection. In macrophages, all intracellular bacteria were cleared within 3 days indicating that persistence of highly virulent strains is restricted to non-professional phagocytes.

2. The percentage of small and SCV phenotypes recovered during the time-course of 7 days was determined as described (Fig 1B) and reaches up to 40% after 7 days for HUVECs and osteoblasts.

3. Changes in bacterial RNA expression for fibronectin binding protein A (fnbA), α-haemolysin (hla) and agr during the course of infection were determined as described (n = 5, ±SD; Fig 1C) and revealed similar changes following infection of different cell types.

4. Changes in host cell response measured by the expression of CCL5, CXCL11 and ICAM-1 (n = 6). *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001 in comparison with values from uninfected cells.

Figure 3. Staphylococci persist in tissues of infected animals while changing phenotypes and virulence factor expression

1. Bacterial loads in the kidneys (triangles) and tibias (circles) of C57BL/6 mice were analysed at progressing times after intravenous inoculation with 5 × 10⁵ CFU of S. aureus 6850. Each point represents the mean ± SD of five mice per group.

2. The percentage of small and SCV phenotypes recovered during the time-course of 28 days was determined as described (Fig 1B).

3. Changes in bacterial RNA expression for fibronectin binding protein A (fnbA), α-haemolysin (hla) and agr during the course of infection were determined by extracting bacteria from bone tissue via magnetic beats, isolating RNA and performing real-time PCR (n = 5, ±SD). The results revealed similar changes as in the in vitro cell culture experiments.

4. Levels of IL-6 (left pane), IL-10 (middle panel) and TNF-α (right panel) in serum of S. aureus-infected mice at progressive times after bacterial inoculation. Each point represents the mean ± SD of five mice per group. *p ≤ 0.05, **p ≤ 0.01 and ***p ≤ 0.001 in comparison with values from uninfected mice.

Figure 4. Staphylococci obtained from clinical specimens revealed changes in phenotype switching and virulence factor expression

We collected six different tissue specimens from S. aureus endovascular, soft tissue and bone infections, including chronic infection courses (see Supplementary Table 2 of Supporting Information). We analysed bacteria directly from the tissue samples and after one subcultivating step in rich medium (BHI, 24 h, shaking).

1. To determine bacterial phenotypes within infected host tissue, we plated homogenized tissue samples on agar plates and defined the percentage of the small and SCV phenotypes. These results were compared to the corresponding bacteria plated after one subcultivating step (n = 6, *p ≤ 0.05).

2. Bacterial RNA expression for fibronectin binding protein A (fnbA), α-haemolysin (hla) and agr was analysed in bacteria directly extracted from two representative documented chronic infections (heart and bone tissue) with magnetic beats. The results were compared to RNA expression in the corresponding bacteria, which were subcultivated for 24 h and grown to the late exponential phase for another 4 h (n = 5, ±SEM, *p ≤ 0.05, **p ≤ 0.01 and ***p ≤ 0.001).

Figure 5. SCVs recovered from infection models and clinical specimens rapidly revert to their fully virulent wild-type phenotype

1. SCV phenotypes recovered from in vitro (cell culture; Fig 1) and in vivo (animals; Fig 3) chronic infection models and from an osteomyelitis patient (A26026V) were subcultivated in BHI at 37°C with shaking and every hour samples were plated on agar plates to determine the percentage of small and SCV phenotypes.

2. Endothelial cells (HUVECs) were infected with different staphylococcal strains and phenotypes, including strain 6850, its isogenic hemB SCV mutant (IIb13), the complemented wild-type mutant (KM4), SCV phenotypes from 6850 recovered after 28 days from in vitro (Fig 1) and in vivo (Fig 2) chronic infection models and clinical SCVs isolated from osteomyelitis patients (A26026V). All SCVs were compared to their corresponding reverted wild-type phenotypes, which were gained by a 24 h subcultivating step in rich medium (BHI).

3. The invasiveness of the different bacterial phenotypes was determined by a flow cytrometric invasion assay.

4. The capacity of the bacteria to induce an acute cytotoxic effect was tested by PI-staining of the endothelial host cells 24 h post-infection (n = 5, ±SEM, **p ≤ 0.01 and ***p ≤ 0.001).