Detection of group A Streptococcus in tonsils from pediatric patients reveals high rate of asymptomatic streptococcal carriage

<p>Abstract</p> <p>Background</p> <p>Group A <it>Streptococcus </it>(GAS) causes acute tonsillopharyngitis in children, and approximately 20% of this population are chronic carriers of GAS. Antibacterial therapy has previously been shown to be insufficient at clearing GAS carriage. Bacterial biofilms are a surface-attached bacterial community that is encased in a matrix of extracellular polymeric substances. Biofilms have been shown to provide a protective niche against the immune response and antibiotic treatments, and are often associated with recurrent or chronic bacterial infections. The objective of this study was to test the hypothesis that GAS is present within tonsil tissue at the time of tonsillectomy.</p> <p>Methods</p> <p>Blinded immunofluorescent and histological methods were employed to evaluate palatine tonsils from children undergoing routine tonsillectomy for adenotonsillar hypertrophy or recurrent GAS tonsillopharyngitis.</p> <p>Results</p> <p>Immunofluorescence analysis using anti-GAS antibody was positive in 11/30 (37%) children who had tonsillectomy for adenotonsillar hypertrophy and in 10/30 (33%) children who had tonsillectomy for recurrent GAS pharyngitis. Fluorescent microscopy with anti-GAS and anti-cytokeratin 8 & 18 antibodies revealed GAS was localized to the tonsillar reticulated crypts. Scanning electron microscopy identified 3-dimensional communities of cocci similar in size and morphology to GAS. The characteristics of these communities are similar to GAS biofilms from <it>in vivo </it>animal models.</p> <p>Conclusion</p> <p>Our study revealed the presence of GAS within the tonsillar reticulated crypts of approximately one-third of children who underwent tonsillectomy for either adenotonsillar hypertrophy or recurrent GAS tonsillopharyngitis at the Wake Forest School of Medicine.</p> <p>Trial Registration</p> <p>The tissue collected was normally discarded tissue and no patient identifiers were collected. Thus, no subjects were formally enrolled.</p

Srv Mediated Dispersal of Streptococcal Biofilms Through SpeB Is Observed in CovRS+ Strains

Group A Streptococcus (GAS) is a human specific pathogen capable of causing both mild infections and severe invasive disease. We and others have shown that GAS is able to form biofilms during infection. That is to say, they form a three-dimensional, surface attached structure consisting of bacteria and a multi-component extracellular matrix. The mechanisms involved in regulation and dispersal of these GAS structures are still unclear. Recently we have reported that in the absence of the transcriptional regulator Srv in the MGAS5005 background, the cysteine protease SpeB is constitutively produced, leading to increased tissue damage and decreased biofilm formation during a subcutaneous infection in a mouse model. This was interesting because MGAS5005 has a naturally occurring mutation that inactivates the sensor kinase domain of the two component regulatory system CovRS. Others have previously shown that strains lacking covS are associated with decreased SpeB production due to CovR repression of speB expression. Thus, our results suggest the inactivation of srv can bypass CovR repression and lead to constitutive SpeB production. We hypothesized that Srv control of SpeB production may be a mechanism to regulate biofilm dispersal and provide a mechanism by which mild infection can transition to severe disease through biofilm dispersal. The question remained however, is this mechanism conserved among GAS strains or restricted to the unique genetic makeup of MGAS5005. Here we show that Srv mediated control of SpeB and biofilm dispersal is conserved in the invasive clinical isolates RGAS053 (serotype M1) and MGAS315 (serotype M3), both of which have covS intact. This work provides additional evidence that Srv regulated control of SpeB may mediate biofilm formation and dispersal in diverse strain backgrounds

Dispersal of Group A Streptococcal Biofilms by the Cysteine Protease SpeB Leads to Increased Disease Severity in a Murine Model

Group A Streptococcus (GAS) is a Gram-positive human pathogen best known for causing pharyngeal and mild skin infections. However, in the 1980's there was an increase in severe GAS infections including cellulitis and deeper tissue infections like necrotizing fasciitis. Particularly striking about this elevation in the incidence of severe disease was that those most often affected were previously healthy individuals. Several groups have shown that changes in gene content or regulation, as with proteases, may contribute to severe disease; yet strains harboring these proteases continue to cause mild disease as well. We and others have shown that group A streptococci (MGAS5005) reside within biofilms both in vitro and in vivo. That is to say that the organism colonizes a host surface and forms a 3-dimensional community encased in a protective matrix of extracellular protein, DNA and polysaccharide(s). However, the mechanism of assembly or dispersal of these structures is unclear, as is the relationship of these structures to disease outcome. Recently we reported that allelic replacement of the streptococcal regulator srv resulted in constitutive production of the streptococcal cysteine protease SpeB. We further showed that the constitutive production of SpeB significantly decreased MGAS5005Δsrv biofilm formation in vitro. Here we show that mice infected with MGAS5005Δsrv had significantly larger lesion development than wild-type infected animals. Histopathology, Gram-staining and immunofluorescence link the increased lesion development with lack of disease containment, lack of biofilm formation, and readily detectable levels of SpeB in the tissue. Treatment of MGAS5005Δsrv infected lesions with a chemical inhibitor of SpeB significantly reduced lesion formation and disease spread to wild-type levels. Furthermore, inactivation of speB in the MGAS5005Δsrv background reduced lesion formation to wild-type levels. Taken together, these data suggest a mechanism by which GAS disease may transition from mild to severe through the Srv mediated dispersal of GAS biofilms

Daily wound irrigation not responsible for the reduction in lesion size observed in E64 treated animals.

<p>Lesion size in saline treated animals (<i>n</i> = 3) was significantly reduced at 5 and 6 dpi compared to MGAS5005Δ<i>srv</i> infected animals (<i>p</i><0.05), however, lesions in saline treated animals were statistically larger than those in E64 treated animals at 2–8 dpi (<i>p</i><0.05).</p

Use of the chemical inhibitor of cysteine proteases E64 significantly reduced lesion size in MGAS5005Δ<i>srv</i> infected animals.

<p>Representative images of lesions formed in mice at 1, 3 and 8 days following subcutaneous infection with ∼2×10⁸ CFU of MGAS5005Δ<i>srv</i>. (A) The infecting dose of MGAS5005Δ<i>srv</i> was suspended in 333 uM E64 (0.1 ml), and lesion development (mm²) was monitored over 8 days (<i>n</i> = 10 mice). A significant reduction in lesion formation was observed when E64 was inoculated with the infecting dose of MGAS5005Δ<i>srv</i> compared to inoculation with MGAS5005Δ<i>srv</i> alone (<i>p</i><0.05). (B) Following inoculation of animals with E64+MGAS5005Δ<i>srv</i> as before, an additional inoculation of 333 µM E64 (0.1 mL) was injected directly into the abscess each day following infection (<i>n</i> = 10). A significant reduction in lesion size was observed with E64 treated animals forming lesions roughly equivalent in size to untreated MGAS5005 infected animals.</p

Immunofluorescent antibody staining revealed detectable levels of SpeB throughout MGAS5005Δ<i>srv</i> infected tissue as compared to MGAS5005 infected tissue.

<p>Subcutaneous abscesses from (A) MGAS5005 and (B) MGAS5005Δ<i>srv</i> infections were excised 1 dpi, sectioned, and stained with rabbit anti-SpeB sera and goat anti-GAS sera, and the appropriate fluorescent secondary antibody conjugate. (A, B) DIC/fluorescent images (4×) from an MGAS5005 infected animal (A) and an MGAS5005Δ<i>srv</i> infected animal (B) show the distribution of GAS (red) throughout the abscess. Randomly selected areas throughout the abscesses were examined for the colocalization of GAS and SpeB (20×, i–iv). MGAS5005 was readily detected (Ai–iv), but SpeB (green) was rarely detected in MGAS5005 infected samples (arrows, Aiii). In contrast, SpeB was detected in the presence of MGAS5005Δ<i>srv</i> throughout the infected samples (Bi–iv). Colocalized SpeB and GAS appear yellow. Representative images are shown. (C) Average total area of pixels (pixels²) was calculated for anti-GAS and anti-SpeB staining in the representative images shown of MGAS5005 and MGAS5005Δ<i>srv</i>. Comparable amounts of anti-GAS staining was observed, however, there is significantly more anti-SpeB staining in MGAS5005Δ<i>srv</i> images compared to MGAS5005 (* <i>p</i><0.01).</p

Histopathology of excised lesions from MGAS5005 and MGAS5005Δ<i>srv</i> infections.

<p>Lesions were surgically excised at days 1, 3, and 8 post infection. 10 µm sections were subjected to H&E staining. Representative low-magnification images (2×) from each time point are shown. (A,B) Infection with MGAS5005 resulted in the formation of a subcutaneous abscess (arrow) that was well delineated by fibrin (pink border) and PMNs (purple border). (C) By 8 dpi, the abscess had ruptured and formed a cutaneous lesion that showed signs of healing (arrow). (D) MGAS5005Δ<i>srv</i> infection resulted in a cutaneous lesion (arrow). (D & E) Note the subcutaneous abscess was less contained by colocalized fibrin and PMNs (dashed arrows). (E) The cutaneous lesion grew in size and did not show any appreciable healing by 8 dpi (F).</p

Bacterial load recovered from excised lesions.

<p>Lesions from mice infected with either MGAS5005 or MGAS5005Δ<i>srv</i> (<i>n</i> = 3 mice/strain) were excised at 1, 3 and 8 dpi, weighed and homogenized for replicate plating. No significant difference in (A) total CFU recovered or (B) CFU/g was observed at 1, 3, and 8 dpi.</p