Viable group A streptococci in macrophages during acute soft tissue infection

Abstract

Background: Group A streptococcal severe soft tissue infections, such as necrotizing fasciitis, are rapidly progressive infections associated with high mortality. Group A streptococcus is typically considered an extracellular pathogen, but has been shown to reside intracellularly in host cells.

Methods and findings: We characterized in vivo interactions between group A streptococci (GAS) and cells involved in innate immune responses, using human biopsies (n = 70) collected from 17 patients with soft tissue infections. Immunostaining and in situ image analysis revealed high amounts of bacteria in the biopsies, even in those collected after prolonged antibiotic therapy. Viability of the streptococci was assessed by use of a bacterial viability stain, which demonstrated viable bacteria in 74% of the biopsies. GAS were present both extracellularly and intracellularly within phagocytic cells, primarily within macrophages. Intracellular GAS were predominantly noted in biopsies from newly involved tissue characterized by lower inflammation and bacterial load, whereas purely extracellular GAS or a combination of intra- and extracellular GAS dominated in severely inflamed tissue. The latter tissue was also associated with a significantly increased amount of the cysteine protease streptococcal pyrogenic exotoxin SpeB. In vitro studies confirmed that macrophages serve as reservoirs for viable GAS, and infection with a speB-deletion mutant produced significantly lower frequencies of cells with viable GAS following infection as compared to the wild-type bacteria.

Conclusions: This is the first study to demonstrate that GAS survive intracellularly in macrophages during acute invasive infections. This intracellular presence may have evolved as a mechanism to avoid antibiotic eradication, which may explain our finding that high bacterial load is present even in tissue collected after prolonged intravenous antibiotic therapy. This new insight into the pathogenesis of streptococcal soft tissue infections highlights a need for alternative therapeutic strategies.

Figure 1. High Bacterial Load and Infiltration of Phagocytic Cells in Tissue Biopsies

Tissue biopsies of clinical grades 1 or 2 (for definitions, see Methods) were stained for the presence of bacteria, macrophages (CD68-positive cells), and neutrophils (neutrophil elastase-positive cells). The stained biopsies were evaluated by ACIAs, and the ACIA value is shown in the figure; for details, see Methods. (A) Percentage of biopsies with low or high bacterial load (indicated by white and black bars, respectively) in biopsies divided according to their clinical grade (1 or 2) or by time of collection after diagnosis. Numerals within the bars denote number of biopsies. Low or high bacterial is defined by the threshold ACIA value of ≥5, as previously described [4]. The viability of GAS in the tissue was assessed by use of a bacterial viability stain that stains viable bacteria green and dead bacteria red, as shown in (B). Note that the viable bacteria are mainly found associated with cells indicated by their red cell nuclei. (C) Degree of neutrophil and macrophage infiltration in biopsies of varying clinical grade. The insets show representative immunohistochemical stainings. (D) Amount of neutrophils (squares) and macrophages (dashed line and circles) in relation to bacterial load, i.e., GAS ACIA. Significant correlation as determined by Deming regression is indicated by p-values.

Figure 2. GAS Were Localized Extracellularly or Intracellularly in Phagocytic Cells in the Tissue

Biopsies from patients with tissue infections (n = 70) caused by GAS were cryosectioned and immunostained for GAS. Immunohistochemical staining revealed three main staining patterns: (A) cytoplasmic, intracellular staining; (B) a combination of intra- and extracellular staining; and (C) extracellular staining. (D) Confocal microscopy of GAS (Alexa 546, red) and CD68-positive cells (Alexa 488, green) in the tissue. The figure shows a simulated fluorescence process projection of a sequential scan. The Y–Z area equals the vertical plane. Intracellular streptococci are shown in yellow. (E) Bacterial localization in relation to clinical grade. Tissue biopsies from patients with tissue infections caused by GAS were classified according to clinical grade (for definitions, see Methods). Numerals within bars denote number of biopsies. Statistical difference between groups was analyzed using Fisher's exact test.

Figure 3. Amount of Spe in Tissue Biopsies with Different Bacterial Localization

Tissue biopsies were immunohistochemically stained for GAS, SpeB, and SpeF, and the stainings were quantified by ACIA. (A) Amount of SpeB and SpeF in biopsies with different bacterial localization (n = 65 and 67, respectively); Intra, intracellular; Intra/Extra, intra and extracellular; and Extra, extracellular. Bars show mean ± SEM. Statistically significant differences were estimated by a Kruskal-Wallis median test. (B) SpeB attached to the bacterial cell surface in a representative tissue biopsy. Confocal microscopy of immunostainings of GAS (Alexa 546, red) and SpeB (Alexa 488, green). Colocalized staining is shown in yellow.

Figure 4. GAS Survive Intracellularly in Monocytes and Macrophages

A) Frequency of cells containing viable intracellular bacteria 4 and 12 h postinfection with strains 8157 and 5448, respectively. Bars denote mean ± SEM of four or five experiments performed using monocytes or macrophages, respectively. (B) A confocal image of a representative macrophage harboring intracellular GAS (viable in green and dead in red) as assessed by staining with a bacterial viability kit. (C) Resurgence of intracellular bacteria (8157 and 5448) in the medium. At 12 h postinfection, antibiotic-free medium was added, and extracellular growth was monitored in the medium over time. The figure shows one representative out of four experiments performed. Control experiments lacking live human cells failed to support growth after antibiotic removal (unpublished data). (D) Comparison of frequencies of cells harboring viable GAS after infection with either 5448 wild-type (WT) strain or its isogenic ΔSpeB knockout (SpeB ko). Infected cells were stained with a live/dead BacLight bacterial viability kit, at 4 and 12 h postinfection, and the frequencies of cells containing viable bacteria in relation to the total number of cells were determined. Mean ± SEM of five experiments performed is shown. Significant difference in survival over time was determined by a Mann-Whitney test, and the p-value is indicated.