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Case Reports
. 2016 Jul 7;1(10):e87882.
doi: 10.1172/jci.insight.87882.

Biofilm in Group A Streptococcal Necrotizing Soft Tissue Infections

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Free PMC article
Case Reports

Biofilm in Group A Streptococcal Necrotizing Soft Tissue Infections

Nikolai Siemens et al. JCI Insight. .
Free PMC article

Abstract

Necrotizing fasciitis caused by group A streptococcus (GAS) is a life-threatening, rapidly progressing infection. At present, biofilm is not recognized as a potential problem in GAS necrotizing soft tissue infections (NSTI), as it is typically linked to chronic infections or associated with foreign devices. Here, we present a case of a previously healthy male presenting with NSTI caused by GAS. The infection persisted over 24 days, and the surgeon documented the presence of a "thick layer biofilm" in the fascia. Subsequent analysis of NSTI patient tissue biopsies prospectively included in a multicenter study revealed multiple areas of biofilm in 32% of the patients studied. Biopsies associated with biofilm formation were characterized by massive bacterial load, a pronounced inflammatory response, and clinical signs of more severe tissue involvement. In vitro infections of a human skin tissue model with GAS NSTI isolates also revealed multilayered fibrous biofilm structures, which were found to be under the control of the global Nra gene regulator. The finding of GAS biofilm formation in NSTIs emphasizes the urgent need for biofilm to be considered as a potential complicating microbiological feature of GAS NSTI and, consequently, emphasizes reconsideration of antibiotic treatment protocols.

Figures

Figure 1
Figure 1. Group A streptococcal (GAS) infections in 3D skin tissue result in biofilm formation.
(A) Quantitative analyses of biofilm formation of clinical GAS necrotizing soft tissue infection isolates on uncoated or fibronectin-coated polystyrene surfaces at indicated time points. The box and whisker plots show median values with min to max distribution (n = 4). (B) Immunofluorescence images of 5626 strain forming biofilm (wheat germ agglutinin [WGA], DAPI, Nile red positive) on uncoated (left panel) and fibronectin-coated (right panel) glass surfaces (original magnification, ×60). Individual stainings are shown in Supplemental Figure 1A. Representative images from 1 of 4 experiments are shown. (C) Representative immunofluorescence images of GAS 800 infected tissue models (original magnification, ×40). GAS is shown in green, and bacterial aggregations are indicated by arrows. Mean fluorescent intensity (MFI) of stained tissue (D), and CFU counts of bacteria recovered from skin models (E). The data represent the mean values ± SD (n ≥ 3). (F) Blinded scoring of tissue pathology of the skin model after infection. Histological severity scoring was performed in a blinded fashion using the following criteria: 0, unaffected; 0.5–1, mild injury with minor epithelial loosening; 1.5–2, moderate injury with some epithelial disruption; 2.5–3, severe injury with continuous epithelial disruption and some detachment; and > 4, extensive injury, massive epithelial disruption, and detachment. Each symbol represents one independent experiment. Horizontal lines denote median values (n = 4). (G) 3D reconstruction of immunostained biofilm in 8157 infected model (original magnification, ×60). GAS-specific antibody, WGA, and Nile red were used. Arrows indicate multilayered bacterial aggregations. (H) SEM images of bacterial biofilm in the skin tissue model 48 hours after infection with indicated clinical isolates (original magnification, ×10,000). The level of significance was determined using 1-way ANOVA with Dunnett’s multiple comparison test.
Figure 2
Figure 2. Biofilm-positive patient biopsies have higher bacterial load and inflammatory responses.
Identification of bacteria in patient biopsies visualized by immunohistochemistry (A) and Gram-staining (B); original magnifications, ×40, and inset figure in B, ×63. (C) Analysis of bacterial load by Gram-scoring. Semiquantitative acquired computerized image analyses (ACIA) of immunohistochemical stainings of group A streptocci (GAS) (D), IL-8 (E), infiltrating neutrophils (F), resistin (G), and HMGB1 (H) in patient biopsies divided based on presence of biofilm or nonbiofilm. Each symbol represents the mean value of the ACIA value in one patient biopsy. The horizontal lines denote the median. The level of significance was determined using 2-tailed Mann-Whitney U test.
Figure 3
Figure 3. Biofilm in tissue biopsies from patients with group A streptococcal (GAS) necrotizing soft tissue infections.
Identification of bacteria in patient biopsies visualized by bacterial viability staining including a 3D reconstruction discriminating viable (green) and dead (red) bacteria (A) (original magnification, ×40). (B) A 3D reconstruction of immunostained biofilm- and non–biofilm-associated patient biopsies with GAS-specific antibody, wheat germ agglutinin (WGA), and Nile red (original magnification, ×63). Arrows indicate multilayered bacterial aggregations. (C) Representative scanning electron micrographs of GAS biofilm and nonbiofilm single-cocci areas in patient biopsies. The boxed area (i) is shown in larger magnification (×10,000) to visualize the biofilm community.
Figure 4
Figure 4. Biofilm formation in skin tissue models infected with group A streptococal (GAS) necrotizing soft tissue infection isolates associated with biofilm or nonbiofilm.
Representative 3D reconstructions of immunofluorescence images of biofilm after 48 hours of skin model infections with strains from patients with biofilm-positive biopsies (2006 and 2028) or biofilm-negative biopsies (5004 and 5006) are shown (original magnification, ×100). GAS-specific antibody, wheat germ agglutinin (WGA), and Nile red were used.
Figure 5
Figure 5. SpeB, capsule, and CovR/S are not associated with biofilm formation in the tissue setting.
(A) Distribution of SpeB-positive and SpeB-negative (SpeB+/SpeB–) clones recovered from biofilm- and non–biofilm-associated patient biopsies. (B) Representative immunofluorescence micrographs of the distribution of SpeB in biofilm and nonbiofilm patient biopsies (original magnification, ×40). (C) SpeB positivity among the necrotizing soft tissue infection strains used for the skin model infections (before infection and 48 hours after). (D) Western Blot analyses of secreted SpeB by indicated strains at different growth stages. Representative images of 3 experiments are shown (n = 3). (E) Relative mRNA expression of speB before and during the infection in tissue models (left panel) or in patient biopsies (right panel; stat., static culture). The data on skin tissue model (left panel) represent the mean values ± SD (n = 3). (F) Amounts of capsular hyaluronic acid in exponential growth phase bacteria. Strains from patients with biofilm-positive or -negative biopsies are shown in circles and squares, respectively. The horizontal line denotes mean values (n = 3).
Figure 6
Figure 6. Gene expression of streptococcal regulatory systems during tissue infections.
(A–D) Relative mRNA expression of genes encoding for streptococcal virulence regulatory systems before and during the infection in tissue models (left panels) or in patient biopsies (right panels; stat., static culture). Relative mRNA expression of genes encoding for regulatory systems mga (A), irr/ihk (B and C), and nra/rofA (D) are shown. The data on skin tissue model (left panel) represent the mean values ± SD (n = 3).
Figure 7
Figure 7. Role of Nra and Sortase A in biofilm formation in the tissue setting.
(A) Amounts of capsular hyaluronic acid in exponential growth phase bacteria. The horizontal line denotes mean values of the 3 experiments. (B) Western Blot analysis of secreted SpeB by indicated strains at late stationary growth stage. Representative image of 3 experiments is shown (n = 3). Original blot is shown in Supplemental Figure 7. (C) CFU counts of indicated bacteria recovered from tissue models after 48 hours of infection. The horizontal line denotes mean values (n = 3). (D) Blinded scoring of tissue pathology of the skin model after infection. Each symbol represents one independent experiment. Horizontal lines denote median values (n = 3). (E) Relative mRNA expression of the transcriptional regulator nra is shown. The data represent the mean values ± SD (n = 3). (F) Representative immunofluorescence images of biofilm after 48 hours of infection (original magnification, ×100). GAS-specific antibody, wheat germ agglutinin (WGA), and Nile red were used.

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