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Ferretti JJ, Stevens DL, Fischetti VA, editors. Streptococcus pyogenes : Basic Biology to Clinical Manifestations [Internet]. Oklahoma City (OK): University of Oklahoma Health Sciences Center; 2016-.
Bacterial infections can occur in almost every part of the human body, which indicates that bacteria have adapted to survive in physiologically distinct anatomical locations. To facilitate this process, an organism must express the proper growth and virulence factors at the appropriate time, endure a potentially harsh surrounding chemical environment, and thwart a host’s immune defenses. Several bacterial species use structures referred to as biofilms to combat these hazards.
A bacterial biofilm is defined as a sessile community of organisms encased in a matrix of extrapolymeric substances and attached to a substratum, interface, or to each other. Biofilms tend to exhibit an altered phenotype with respect to growth rate and gene transcription (Donlan & Costerton, 2002). The development of biofilms can be described by a five-stage process (Sauer, Camper, Ehrlich, Costerton, & Davies, 2002; Stoodley, Sauer, Davies, & Costerton, 2002). Briefly, Stage 1 consists of planktonic cells transiently adhering to a surface. At this stage, only small amounts of extrapolymeric components are associated with the attached cells and many cells are still capable of independent movement (O'Toole & Kolter, 1998). During Stage 2, cells begin to produce larger amounts of extracellular polymer, which leads to a more stable attachment. Stages 3 and 4 involve the establishment and maturation of biofilm architecture. Cell clusters interspersed with water channels form three-dimensional structures that are widely recognized today as microcolonies, and cells within these microcolonies begin to alter their physiology. Stage 5 is associated with the dispersal of individual cells or pockets of cells from the biofilm structure. These cells are free to disseminate, recolonize, and repeat the cycle of biofilm development. In Streptococcus pyogenes, a mature biofilm is known to consist of proteins, DNA, and a polysaccharide-containing material known as glycocalyx (Doern, et al., 2009; Akiyama, Morizane, Yamasaki, Oono, & Iwatsuki, 2003; Cho & Caparon, 2005). (Figure 1)
Biofilms are responsible for a large medical burden throughout the world. According to the US National Institute of Health, biofilms account for over 80% of microbial infections in the human body (Davies, 2003). An estimated 17 million new biofilm infections arise annually in the United States, which result in as many as 550,000 fatalities each year (Worthington, Richards, & Melander, 2012) and cause an ever-growing economic burden, due to chronic infections and longer hospital stays. Biofilms pose a significant health risk because they are inherently tolerant to host defenses and are up to a thousand times more resistant to conventional antibiotics (Rasmussen & Givskov, 2006). Additionally, biofilms formed within medical devices such as prosthetic heart valves, intrauterine devices, central venous catheters, and urinary catheters can be very difficult to eliminate. Their removal requires the use of aggressive antibiotic therapies, surgical debridement, and removal of the infected device (Donlan & Costerton, 2002; Stewart & Costerton, 2001). Biofilm-residing bacteria, including S. pyogenes, are able to persist on both biotic and abiotic surfaces (including soft toys, books, cribs, and other hard surfaces) for extended periods of time. This results in an increased chance of exposure from contact with surfaces that were previously disregarded as a source of transmission (Marks, Reddinger, & Hakansson, 2014a).
Streptococcus pyogenes (group A streptococcus) is a Gram-positive pathogen that is responsible for a wide variety of human disease. Diseases range from relatively mild clinical illnesses such as pharyngitis, cellulitis, and impetigo, to life-threatening conditions, such as puerperal sepsis, myositis, toxic shock syndrome, and necrotizing fasciitis (flesh-eating disease). S. pyogenes infections can also lead to post-infectious sequelae, such as acute rheumatic fever, rheumatic heart disease, and acute post-streptococcal glomerulonephritis (Bisno, Brito, & Collins, 2003; Cunningham, 2000). S. pyogenes has been shown to form biofilms both in vitro and in vivo. Although biofilm production and regulation have been well studied in organisms such as Pseudomonas aeruginosa and Staphylococcus species, much less is known about S. pyogenes biofilms and their contributions to human disease.
Some of the first images of S. pyogenes biofilm associated with mild human disease were obtained by Akiyama et al. from infected impetigo skin lesions (Akiyama, Morizane, Yamasaki, Oono, & Iwatsuki, 2003). Using confocal laser scanning microscopy (CLSM), S. pyogenes microcolonies were visualized in infected human tissue, which indicates that S. pyogenes residing in a biofilm state may influence impetigo disease pathogenesis. A similar phenomenon was observed in a murine model of skin infection. Murine skin irritated and inflamed by the addition of croton oil resulted in the formation of an S. pyogenes microcolony at the site of infection. This occurred even when mice were orally treated with the antimicrobial agent cefdinir (CFDN). S. pyogenes microcolony formation in the presence of CFDN suggests that biofilm formation affords some level of protection as compared to their planktonic cells, an observation noted from research on other biofilm-producing organisms.
Recent literature suggests that S. pyogenes biofilm formation may be important for maintaining an asymptomatic carriage state in the human tonsil (Roberts, et al., 2012). With tonsils excised from patients suffering from adenotonsillar hypertrophy (ATH) or recurrent S. pyogenes pharyngitis, Roberts et al. were able to use immunofluorescence microscopy to visualize S. pyogenes microcolonies within tonsillar reticulated crypts (Roberts, et al., 2012). Three-dimensional bacterial communities arranged in biofilm-like structures were also visible in scanning electron microscopy (SEM) images. Because up to one third of pharyngitis cases don’t respond to antimicrobial treatment (Kuhn, et al., 2001; Macris, et al., 1998), the presence of S. pyogenes biofilm in tonsillar crypts may demonstrate one mechanism for the increased survival of this microorganism during antimicrobial therapies. Roberts et al. (Roberts, Connolly, Doern, Holder, & Reid, 2010) studied S. pyogenes biofilm pathogenesis in a chinchilla middle-ear model of otitis media. Hemotoxylin and Eosin (H&E) and Gram stains were used to analyze macroscopic structures removed from the middle ear cavity of chinchillas that had been infected with S. pyogenes by intrabullar injection. S. pyogenes microcolonies were observed with both staining techniques, indicating their ability to colonize the middle ear cavity. SEM analysis also revealed the presence of three-dimensional biofilms in the removed macroscopic structures (Roberts, Connolly, Doern, Holder, & Reid, 2010).
In work designed to mimic periapical periodontitis, Takemura et al. (Takemura, et al., 2004) analyzed the ability of S. pyogenes to form biofilms on gutta percha points, the material used in tooth cavity repair and root canal procedures. In dental practice, S. pyogenes is routinely isolated from infected root canal (Le Goff, Bunetel, Mouton, & Bonnaure-Mallet, 1997). With the use of SEM, S. pyogenes biofilms were seen on the surface of infected gutta percha points, which demonstrates their ability to colonize a clinically relevant abiotic surface.
The contribution of S. pyogenes biofilm to cellulitis was explored by Connolly et al. using a murine model of soft tissue infection (Connolly, Roberts, Holder, & Reid, 2011a). Following subcutaneous infection with S. pyogenes, excised sections of infected murine tissue were processed for the presence of microcolony formation. Gram staining of the excised sections, as well as immunofluorescence microscopy, revealed microcolony formation within the tissue.
Neely et al. (Neely, Pfeifer, & Caparon, 2002) used zebrafish to study S. pyogenes pathogenesis associated with myositis. In H&E stained and immunofluorescent images of infected zebrafish muscular tissue, S. pyogenes was present in large, densely packed clusters of bacteria. There was also a marked absence of inflammatory cell infiltration at the site of tissue damage. This disease pathology was also previously seen in a baboon model of fatal intramuscular streptococcal infection (Taylor, et al., 1999).
S. pyogenes biofilms may also play a role in the severity and progression of life-threatening human disease. Hidalgo-Grass et al. (Hidalgo-Grass, et al., 2004) used H&E staining to visualize S. pyogenes cell clusters in surgically debrided tissue sections from two patients suffering from necrotizing fasciitis (flesh-eating disease) and myonecrosis. Tissue sections were characterized by large amounts of S. pyogenes cells and a lack of neutrophil infiltration.
Notably, data suggests that biofilm-residing S. pyogenes colonizing nasal-associated lymphoid tissue (NALT) in mice are naturally transformable, as compared to planktonic bacteria that are typically non-competent. The extent to which this contributes to S. pyogenes genetic variation in vivo is unknown (Marks, Mashburn-Warren, Federle, & Hakansson, 2014b).
M proteins are well characterized virulence factors and are the major determinants for S. pyogenes serotyping, with over 200 serotypes currently categorized (Cunningham, 2000; Cole, Barnett, Nizet, & Walker, 2011). The primary M-protein used for serotyping is encoded by the emm gene, which is found within the mga regulon (Courtney, et al., 2009). This family includes the M protein (Emm), M-related protein (Mrp), and an M-like protein (Enn). Some S. pyogenes serotypes can encode just the emm gene, while others possess a combination of emm, enn and mrp (Bessen, Izzo, McCabe, & Sotir, 1997; Bessen, Sotir, Readdy, & Hollingshead, 1996; Kalia & Bessen, 2004). This family of cell surface proteins has been shown to play a role in S. pyogenes biofilm formation.
Lembke et al. were interested in characterizing biofilms produced by clinically relevant serotypes of S. pyogenes. These authors studied one isolate from nine different clinically relevant S. pyogenes serotypes (M1, M2, M3, M6, M12, M14, M18, M28, and M49) to assess primary adhesion to varying surfaces. These surfaces included uncoated and coated plastic surfaces with fibronectin, fibrinogen, collagen types I and IV, and laminin. Variation was observed among the different serotypes, with M2, M6, M14, and M18 adhering to the different host matrix protein-coated surfaces in measurable amounts. Serotype strains M1, M12, M28, and M49 had little primary adherence to both uncoated or matrix protein-coated surfaces (Lembke, et al., 2006). Lembke et al. suggested that the strains tested from serotypes M1, M12, M28, and M49 are unable to form potential biofilms. Serotype strains M2 and M6, and to a lesser extent those from M14 and M3, were able to transition from planktonic to sessile cells on fibronectin- and fibrinogen-coated surfaces. Adhesion was only supported on collagen I, collagen IV, and lamin for M2 after 72 hours of incubation. The M18 serotype isolate only adhered to collagen type I- or IV-coated surfaces. Since M6 and M14 strains could adhere to any of the matrix protein-coated surfaces, as well as the uncoated plastic surface, the authors suggested that these isolates qualified as potential biofilm builders. The biofilm formation behavior was investigated between different isolates of the serotypes M1, M3, and M6, and revealed heterogeneity amongst the strains. Lembke et al. suggested that biofilm formation is a trait of the individual S. pyogenes isolate, rather than of the defined S. pyogenes serotypes (Lembke, et al., 2006).
These isolates were further studied to determine if they were able to form typical biofilm architectures. Safrinin staining was used to visualize the biofilm architecture of S. pyogenes isolates after 72 hours of incubation on plastic coverslips coated with their preferred substrates. Serotypes M6 and M14 were able to adhere to all surfaces, including uncoated polystyrene, whereas serotype M2 bound to fibronectin, fibrinogen, lamin, and collagen types I and IV. Binding was only seen for serotype M2 on lamin and collagen types I and IV after 72 hours. Serotype M18 bound exclusively to collagen types I and IV. The strains from serotypes M1, M12, and M49 were unable to form dense layers on any substrate tested, which is consistent with their inability to gain measurable amounts of primary adherence on these substrates. Scanning electron microscopy (SEM) was performed to confirm that the structures seen using light microscopy were in fact biofilm-like structures. A S. pyogenes strain from serotype M49 displayed few bacterial chains on the fibronectin-coated surface, but no multilayered meshwork after 72 hours of incubation. The M6 isolate grown on plastic-coated surfaces and the M18 isolate grown on collagen type IV-coated surfaces both formed three-dimensional, multilayered, dense biofilms. Increased resolution revealed the presence of a multilayer structure, which consisted of “secondary colonizers” adhering to the apical surface of the primary attached layer. Although typical biofilm structures could be revealed through SEM, no extrapolymeric substance (EPS) was detected for the biofilm-positive S. pyogenes serotype strains under the conditions tested (Lembke, et al., 2006).
Confocal laser scanning microscopy (CLSM) was then used to determine the depth of biofilm. The S. pyogenes isolates from serotypes M2, M6, and M18 were transformed with a plasmid that constitutively expressed egfp (enhanced green fluorescent protein gene), which created strains TM2, TM6, and TM18, respectively. Three-dimensional structures of 72-hour biofilms of TM2, TM6, and TM18 had thicknesses measuring 13.3, 13.6, and 28 microns, respectively. Additionally, Lembke et al. calculated that these biofilms contained 13–46 cell layers by using the assumptions that the S. pyogenes cell diameter ranges from 0.6 to 1 microns. The state of the biofilm was assessed over extended incubations of 72, 96, and 120 hours. At 72 hours, M18 reached maximum attachment. After 96 hours, serotype strains M2 and M6 reached peak biofilm formation, but longer incubations resulted in decreased attachment. 120 hours of incubation of serotype strain M2 resulted in partial disintegration of the biofilm, as well as significantly shorter chains (Lembke, et al., 2006).
Lembke et al. suggested that morphological changes occur in S. pyogenes biofilms during long incubation times. S. pyogenes strains from serotypes M2 and M18 were analyzed under continuous flow conditions, and were found to form biofilms within this chamber system. Using SEM, the M18 isolate was found to form biofilms; however, these were not as dense as those observed in static conditions. These biofilms were also oriented in the direction of the medium flow and had no EPS material present. The M2 isolate had a more compact biofilm structure under flow conditions, as compared to the M18 strain, and also had chains connected by threadlike structures of unknown chemical composition (Lembke, et al., 2006).
A later study by Thenmozhi et al. was interested in characterizing biofilms of different clinical S. pyogenes M serotypes (Thenmozhi, Balaji, Kumar, Rao, & Pandian, 2011). Eleven serotypes were grown in Todd Hewitt broth (THB) for 24, 48, 72, and 96 hour intervals to observe biofilm formation. Crystal violet staining was used to quantitate the biofilms and CLSM was used to calculate surface area and thickness. Five of the 11 serotypes tested in this study did not form any biofilms and were classified as non-biofilm formers (M49, M63, M88.3, M122, and st2147). Six of the serotypes (M56, M65, M74, M89, M100, and st38) formed substantial biofilms, comparable to the positive control (Streptococcus mutans UA159, a known biofilm former). These strains formed significant biofilms at 48 hours with gradual reduction at later time intervals. However, serotype strain M65 showed significant biofilm formation at 96 hours, as compared to the other time points. Overall, variation was observed among the biofilm formers at the various time points. Serotype M56 alone produced copious amount of biofilms at all the time points (Thenmozhi, Balaji, Kumar, Rao, & Pandian, 2011).
Biofilm-forming serotypes showed maximum surface area coverage at 48 hours, except for st38. Serotype strain M56 formed dense biofilms, as compared to the other serotypes, and had a maximum of 35% surface area coverage. Thickness between serotypes was independent of the time intervals. Serotype strains M56 and M100, which formed the largest biofilms, had thicknesses measuring 8.2 to 6.8 microns, respectively (Thenmozhi, Balaji, Kumar, Rao, & Pandian, 2011).
Oliver-Kozup et al. were interested in studying the biofilm formation of strains from multiple serotypes of S. pyogenes (M1: MGAS5005, M3: MGAS315, M28: MGAS6143, and M41: MGAS6183) (Oliver-Kozup, et al., 2011). Each of these strains was assessed for its ability to form biofilms using the crystal violet staining method, followed by CLSM and SEM. After 24 hours, M41 and M28 serotypes produced the largest biomass, as compared to M1 and M3, as assessed by crystal violet staining. Serotype M3 did not produce a substantial biomass, as compared to the other serotypes. Oliver-Kozup et al. suggested that these findings indicate a variation among S. pyogenes strains in their ability to form biofilms in vitro (Oliver-Kozup, et al., 2011).
CLSM was performed to visualize biofilm formation of three M3 serotype strains (MGAS315, MGAS2079, and MGAS158). The CLSM results confirmed what was seen in the crystal violet assay: a deficiency in the ability to form any appreciable biofilm structure. M41, M28, and M1 serotypes were capable of forming appreciable biofilms. After 24 hours, the average thickness was found to differ among all three serotypes, in a manner similar to the crystal violet data. Serotype M41 produced the thickest biofilms at 15 microns with M28 and M1 producing thinner biofilms at 12 microns and 9 microns, respectively. Additionally, biofilm cross-sections revealed architectural differences among the three serotypes. The thickest biofilms (M41 and M28), were less dense but had more elevated supracellular assembly. Serotype M1, the thinnest biofilm, was made of densely-packed cells that formed continuous layers. Field emission scanning electron microscopy (FESEM) was used to gain more insight on the architecture of the thinnest and thickest biofilms (M1 and M41). The M41 serotype biofilm had a more diverse architecture, with depressions and crypts, as compared to serotype M1. Additionally, the M41 cells had a studded cell surface morphology with protrusions that linked both adjacent cells and chains. Alternatively, M1 biofilms lacked pronounced surface characteristics and had a more smooth appearance with a rich bacterial-associated extracellular matrix (BAEM). BAEM was not observed in the biofilm of the M41 serotype. To further compare the production of BAEM between the biofilms of the M1 and M41 strains, green-fluorescent protein (GFP) expressing strains were created. TRITC-concanavalin A (ConA), a fluorescently-labeled lectin that binds to the extracellular polysaccharides in biofilms (Maeyama, Mizunoe, Anderson, Tanaka, & Matsuda, 2004), was used to stain serotype M1 and M41 biofilms grown for 24 hours on glass coverslips. Fluorescent microscopy was performed to visualize the BAEM material. The results showed that the M1 strain had a dense and closely associated matrix. Oliver-Kozup et al. suggested that the stability of S. pyogenes biofilms differs among serotype strains and that more BAEM production does not necessarily pre-determine a larger biofilm mass (Oliver-Kozup, et al., 2011).
A study by Cho and Caparon investigated whether virulence factors, such as M protein, were crucial for biofilm formation; they observed biofilm formation of the wild-type M14 serotype strain HSC5 (with a mutagenic plasmid integrated at an intergenic locus) and HSC5 strain with an insertional disruption of the emm gene. Using a microtiter plate assay, biofilms were grown in a peptide rich, but carbohydrate poor C medium at 23°C for 24, 48, 72, and 96 hours (Cho & Caparon, 2005). The HSC5 strain with a disruption in the emm gene was unable to form biofilms at any of the time intervals. Because this assay was used to detect the initial cell-surface interactions required for biofilm formation, Cho and Caparon suggest that this data indicates that M protein influences the initial stage in biofilm development. Additionally, growth in flow chambers was assessed under the same conditions using the wild-type and disrupted emm gene strain of HSC5. Similar to the microtiter assay, a disruption in the emm gene resulted in the inability to form biofilms under flow conditions. Cho and Caparon indicate that based on this data, M protein is important for initial cell-surface interactions in biofilm development (Cho & Caparon, 2005).
To test the dependence of surface proteins on biofilm formation Courtney et al added trypsin to S. pyogenes growth media (Courtney, et al., 2009). Trypsin cleaves surface proteins, but does not affect the growth of S. pyogenes. All strains tested were unable to form biofilms. Therefore, Courtney et al. suggest that S. pyogenes biofilm formation is dependent on streptococcal surface proteins. To determine the role of the various members of the M protein family (Emm, Mrp, Enn, and Spa) on biofilm formation M protein family mutants were compared to their specific wild-type for their ability to form biofilms. In serotypes with only one emm gene that encodes M protein (M1, M5, M6, and M24), inactivation resulted in a deficiency in biofilm formation. Serotypes with multiple genes that encode M proteins were shown to have varying effects when individual genes were inactivated. Inactivation of emm2 in an M2 strain had a minor effect on biofilm formation, with the inactivation of mrp2 having no significant effect. mrp4 in an M4 strain resulted in a 96% reduction in biofilm formation relative to wild-type, and inactivation of emm4 and enn4 had no effect. Reductions of 30% and 50% were observed with inactivation of emm49 and mrp49, respectively in serotype strain M49. In serotype M18, inactivation of Spa (streptococcal protective antigen surface protein) resulted in a decrease of biofilm formation by 42%, as compared to wild-type M18 (Courtney, et al., 2009).
A later study by Courtney et al. sought to determine which members of the M protein family are involved in S. pyogenes biofilm formation. More specifically, researchers were interested in whether these M protein family members were anchoring lipoteichoic acid (LTA) in a manner that contributes to hydrophobicity. LTA has been known to contribute to the hydrophobicity of Gram-positive bacteria (Doyle & Rosenberg, 1990; Miörner, Johansson, & Kronvall, 1983; Fedtke, et al., 2007). Hydrophobicity of several S. pyogenes serotypes is dependent on the expression of surface proteins that form complexes with LTA, such that the ester linked fatty acids of LTA are exposed on the S. pyogenes surface (Courtney, et al., 2009; Ofek, Whitnack, & Beachey, 1983).
Biofilm formation was studied using mutant strains from serotypes M1, M2, M4, M5, M6, M18, M24, and M49, which were constructed through allelic replacement of specific genes that encode the M protein(s). The addition of glucose to tryptic soy broth (TSB) had a minor effect on planktonic growth, but had a three-fold increase on biofilm formation. However, Todd-Hewitt broth plus yeast (THY) was the best medium for growth (as well as for biofilm formation), and was chosen as the standard for the remaining studies in this area. Variation in biofilm formation was observed among all serotypes. Serotypes M2 and M6 had the highest degree of biofilm formation, while serotype M49 had the least degree of biofilm formation (Courtney, et al., 2009).
Courtney et al. showed that M proteins are involved in biofilm formation, hydrophobicity, and adhesion to hexadecane. A competitive inhibition enzyme-linked immunosorbent assay (ELISA) revealed that M proteins play a role in the amount of protein-bound LTA. To determine if the levels of M protein could have an effect on hydrophobicity and biofilm formation, Courtney et al. constructed a recombinant strain that overexpresses emm1, with a two-fold increase in Emm1 production in the M1 serotype. Results indicated that increased production of Emm1 increases the amount of protein-bound LTA, hydrophobicity, and biofilm formation, as compared to the wild-type strain and emm1 inactivated strain. Courtney et al. suggested that the formation of complexes between M proteins and LTA directly contributed to both hydrophobicity and biofilm formation in most S. pyogenes serotypes; however, in some serotypes, a direct link could not be demonstrated. These authors also suggested that the absence of a direct link might be attributed to some serotypes that possess multiple M protein family members and that the inactivation of a single member is not sufficient to alter these functions (Courtney, et al., 2009).
Pili are long filamentous structures found on the surface of several bacterial species, including S. pyogenes (Mora, et al., 2005). S. pyogenes pilus-associated proteins are encoded on a pathogenicity island named the fibronectin-binding, collagen-binding, T-antigen (FCT) region (Bessen & Kalia, 2002). This region contains genes that code for pilus structural subunits and for the sortase enzymes required for pilus assembly (Bessen & Kalia, 2002; Manetti, et al., 2007). In specific S. pyogenes strains, multiple components of pili assembly and structure have been identified for their role in biofilm formation. These include srtA, which encodes a housekeeping sortase, the T shaft protein (tee gene), and Ancillary Protein 1 (AP1) (Becherelli, et al., 2012; Nakata, et al., 2011; Kratovac, Manoharan, Luo, Lizano, & Bessen, 2007). This locus is highly variable, with 9 FCT variants having been identified (Kratovac, Manoharan, Luo, Lizano, & Bessen, 2007; Falugi, et al., 2008). The role of pili in the S. pyogenes biofilm life cycle is becoming ever more appreciated (Manetti, et al., 2007; Becherelli, et al., 2012; Köller, et al., 2010; Manetti, et al., 2010; Nakata, et al., 2009; Kimura, et al., 2012).
Evaluation of 24-hour biofilms grown in the peptide-rich, but carbohydrate-poor C medium C-medium by Manetti et al. demonstrated the importance of pili to the maturation of the biofilm structure (Manetti, et al., 2007). A wild-type S. pyogenes serotype M1 strain SF370 (FCT type 2) was five to six times more efficient at generating biofilms on polystyrene than isogenic pilus backbone and sortase C1 mutants. Confocal microscopy of S. pyogenes growing on polylysine-coated coverslips revealed that after 72 hours, strain SF370 was able to form biofilms with an average thickness of 10.8um while the two pili mutants were unable to form a significant multilayered biofilm structure. Manetti and colleagues concluded that one of the main roles of pili is to allow S. pyogenes to switch from planktonic to biofilm growth.
Nakata et al. investigated the effects of pilus-associated protein deletions on biofilm formation using S. pyogenes serotype M49 strain 591 (FCT type 3) (Nakata, et al., 2009). Various FCT-3 component deletion strains (cpa operon and single component mutants: Δcpa, ΔfctA, ΔfctB, ΔsrtC2, and ΔlepA), as well ΔprtF2 and a strain lacking srtA, were tested for their abilities to form static biofilms in C-medium on polystyrene plates at 28°C and 37°C. Results indicated that strains lacking srtA produced significantly less biofilm, regardless of the temperature used. All other mutants produced wild-type levels of biofilm under both conditions, which led Nakata and colleagues to suggest that although srtA is required for biofilm formation of strain 591, srtC2 and other components of the cpa operon do not play a significant role.
Koller et al. evaluated the relationship between FCT type and biofilm formation (Köller, et al., 2010). 183 S. pyogenes isolates (representing pharyngitis, skin infection, and invasive disease) were obtained from University Hospital in Rostock, Germany, from 2001–2006 and were subjected to 24-hour static biofilm assays in C-medium and brain-heart infusion (BHI). Crystal violet analysis of biofilm mass revealed that FCT type 1 strains formed robust biofilms in both culture media; FCT type 2, 5, and 6 formed robust biofilms in BHI broth, but weak biofilms in C-medium; FCT type 9 strains formed weak biofilms in either culture media; FCT type 3 and 4 strains exhibited heterogeneous biofilm growth within their FCT groups in both media. Koller and colleagues concluded that FCT typing represents an additional method for characterization of S. pyogenes and that biofilm growth in defined media may represent a novel epidemiological marker (Köller, et al., 2010).
Manetti et al. established a link between acidic environmental conditions and pilus-mediated S. pyogenes biofilm formation (Manetti, et al., 2010). Using 44 clinical isolates obtained from University Hospital in Rostock, Germany, from 2001–2006, static biofilm assays were carried out in C-medium or C-medium supplemented with 30mM glucose. FCT type 1 strains formed robust biofilms in both culture media; FCT type 2, 3, 5, 6 and a subset of type 4 strains exhibited increased biofilm formation in supplemented media; and FCT type 9 and a subset of type 4 strains failed to form biofilms in either condition. Manetti and colleagues reasoned that a pH decrease in the media due to sugar metabolism was the cause of the glucose-mediated biofilm formation results. They addressed this possibility by performing 12-hour biofilm assays in unbuffered C-medium at either pH 6.4 or 7.5. Results indicated that FCT type 1 strains formed biofilms at both pH levels; FCT type 2, 3, 5, 6, and a subset of type 4 strains showed increased biofilm when grown in media with lower starting pH; FCT type 9 and a subset of type 4 strains were poor biofilm formers in both culture conditions. Manetti and colleagues also demonstrated that pH-dependent biofilm formation was directly associated with differential pilus expression, which led them to conclude that most S. pyogenes FCT type strains sense environmental pH as a signal to build pili on their surface, and that this process may lead to biofilm formation (Manetti, et al., 2010).
The role of the FCT type 1 pili in S. pyogenes biofilm formation was investigated by Kimura et al. using pilus-associated gene deletions (Kimura, et al., 2012). Pilus-associated deletions (Δtee6, ΔfctX, ΔsrtA, ΔsrtB) were created in a S. pyogenes serotype M6 strain TW3558 and the effects on static 24-hour biofilm formation in C-medium were examined. Crystal violet analysis revealed that deletion of pilus-associated proteins resulted in decreased biofilm formation, as compared to wild-type levels. These results were confirmed with confocal microscopic analysis. To determine if the pilus mutant phenotype was present in other M6 strains, tee6 deletions were made in strains SE1303, S43, SE1387, and 97A-85 (Kawabata, et al., 1999; Murakami, et al., 2002). Static biofilm analysis demonstrated that tee6 deletions in strains SE1303, S43, and SE1387 yielded reduced biofilm formation. Therefore, Kimura and colleagues concluded that pili composed of T6 shaft protein are crucial for the biofilm formation process in M6 strains (Kimura, et al., 2012).
Becherelli et al. evaluated how ancillary protein 1 of FCT type 1 pili mediates S. pyogenes biofilm formation (Becherelli, et al., 2012). S. pyogenes serotype M6 strain HRO-27_M6 (obtained from University Hospital in Rostock, Germany) and its isogenic Δap1_M6 mutant were subjected to 10-hour static biofilm assays in C-medium on polystyrene. Deletion of ancillary protein 1 resulted in reduced biofilm formation when compared to wild-type levels. Additionally, incubation of wild-type cells with polyclonal antibodies raised against rAP1_M6 abolished biofilm formation. The data obtained lead Becherelli and colleagues to hypothesize that AP1 pilus components mediate tissue colonization through the formation of large cell-adhering microcolonies (Becherelli, et al., 2012).
It should be noted that several reports have examined the role of S. pyogenes pili in host cell adherence (Abbot, et al., 2007; Crotty Alexander, et al., 2010; Edwards, et al., 2008; Smith, et al., 2010). Because these reports did not specifically investigate biofilm formation, their details have been omitted from this review.
The hyaluronic acid capsule is a major virulence determinant of S. pyogenes (Cunningham, 2000). It is required for resistance to phagocytosis (Wessels & Bronze, 1994) and is an important adherence factor in the pharynx because of its ability to bind CD44 on host epithelial cells (Schrager, Albertí, Cywes, Dougherty, & Wessels, 1998). The capsule is composed of a polymer of hyaluronic acid that contains repeating units of glucuronic acid and N-acetylglucosamine (Stoolmiller & Dorfman, 1969). Synthesis of the polymer involves gene products of the has operon (Dougherty & van de Rijn, 1992) and csrR. CsrR is a regulator in the two-component system CsrRS (also known as CovRS) and has been shown to be a negative regulator of the capsule synthesis process (Levin & Wessels, 1998). The role of the hyaluronic acid capsule in S. pyogenes biofilm formation is still not fully understood.
Cho et al. investigated the importance of the S. pyogenes capsule on biofilm maturation by evaluating the role of the hasA gene. The hasA gene (coding for hyaluronate synthase, the first gene in the has operon) was deleted in serotype M14 strain HSC5, and both static and continuous flow biofilm analyses were conducted. Static biofilm experiments with C-medium at 23°C revealed that a strain that lacked a hyaluronic acid capsule was capable of attaching to a polystyrene substrate. However, continuous flow biofilm analysis revealed that adherent HSC5ΔhasA cells remained as an unorganized layer, rather than forming the typical three-dimensional biofilm structure. These results led Cho and colleagues to conclude that the hasA mutant was fully competent for adherence to the substrates tested, but was unable to progress through the subsequent stages of biofilm maturation. Notably, the covR mutant, which overproduces capsules, was also incapable of forming biofilms under the conditions tested (Heath, DiRita, Barg, & Engleberg, 1999).
Sugareva et al. also examined the effects capsule synthesis has on S. pyogenes biofilm formation (Sugareva, et al., 2010). Static biofilm formation on noncoated, fibronectin-coated, or human collagen type 1-coated plastic coverslips in BHI media supplemented with glucose was evaluated for a panel of strains that represented serotypes M2, M6, M18, and M49. Deletion of covS (a sensor kinase involved in regulation of hyaluronic acid synthesis) in these clinical isolates revealed that effects on biofilm formation appear to be both serotype- and strain-specific. As examples, covS deletions in serotype M18 strains resulted in decreased biofilm formation, whereas two of the serotype M6 strains examined exhibited increased biofilm formation, as compared to wild-type levels. It should be noted that in all strains tested, the amount of capsule detected in covS mutants was increased in comparison with the wild-type parental strain, which indicates that capsular effects on S. pyogenes biofilm formation may be strain-specific.
Zhang et al. sequenced the genome of serotype M28 S. pyogenes strain MGAS6180 (Green, et al., 2005) and identified and characterized M28_Spy1325. M28_Spy1325 is denoted as AspA, for A Streptococcus surface protein A. AspA is a member of the antigen I/II (AgI/II)-family of polypeptides (Brady, et al., 2010; Zhang, Green, Sitkiewicz, Lefebvre, & Musser, 2006), which are important cell surface-anchored molecules produced by oral streptococci (Jenkinson & Demuth, 1997). AgI/II proteins are structurally complex, multifunctional adhesins that bind human salivary glycoproteins and assist in colonization of the oropharynx (Jenkinson & Demuth, 1997; Jakubovics, Strömberg, van Dolleweerd, Kelly, & Jenkinson, 2005). Furthermore, AspA has been shown to play a role in mediating adherence and biofilm formation in group A streptococci (Maddocks, et al., 2011).
Maddocks et al. used two serotype M28 strains, MGAS6180 and H360, to study the role of AspA in biofilm formation on a salivary pellicle. Allelic replacement was used to delete the entire coding region of the aspA gene and insert a spectinomycin antibiotic cassette (aad9) in these strains. The ∆aspA mutants of both strains showed identical colony morphology, growth rates in minimal C medium, and adherence levels to immobilized salivary agglutinin glycoprotein (gp-340), as compared to their respective parent strains. When Maddocks et al. grew biofilms on salivary pellicle-coated cover slips, their results indicated a significant difference in biofilm formation between the ∆aspA mutants and their wild-type strains. After 24 hours of incubation, wild-type strains MGAS6180 and H360 formed biofilms approximately 25µm thick that consisted of densely packed cells with a structure that covered almost the entire underlying salivary pellicle. Both ∆aspA mutants from each strain showed reduced biofilm thickness, visibly revealing much of the salivary pellicle substratum. MGAS6180 ∆aspA had a 60% reduction in biomass, while H360 ∆aspA had a more severe defect in biofilm formation, with a greater than 80% reduction when compared to the H360 parent strain. ∆aspA mutants complemented in trans had restored biofilm formation, but cells were more loosely packed and the architecture appeared more disorganized, as compared to the respective wild-type strains. Maddocks et al. suggested that in these two particular M28 strains, AspA production is essential for biofilm formation (Maddocks, et al., 2011).
In order to define the specificity of AspA in biofilm formation, Maddocks et al. studied the ability of wild-type and ∆aspA mutants to form biofilms on various substrata, including gp-340, 10% saliva-coated polystrene, and polystrene surfaces. Biofilm formation of the wild-type strains MGAS6180 and H360 was similar among the three surfaces. However ∆aspA mutants for both strains showed a reduction in biofilm biomass by 60% when grown on gp-340, and a 50% reduction when grown on 10% saliva-coated polystyrene. There was no significant decrease in biomass when grown on polystyrene-only surfaces between mutant and wild-type strains. When ∆aspA mutants were complemented in trans and grown on these various substrata, they showed biofilm biomasses relative to wild-type levels. Maddocks et al. suggested that AspA has a specificity to the salivary glycoprotein substratum (Maddocks, et al., 2011).
Maddocks et al. further confirmed a role for AspA in promoting adherence and biofilm formation by expressing AspA in Lactococcus lactis strain MG1363. L. lactis wild-type and AspA-expressing strain were grown on salivary pellicle-coated coverslips for 24 hours. L. lactis expressing AspA produced a densely packed biofilm 26µm thick, which was three-fold higher in biomass, as compared to the L. lactis wild-type strain that produced a sparse biofilm (Maddocks, et al., 2011).
The extracellular protein Streptococcal collagen-like protein 1 (Scl1) is encoded by the scl1 gene and has been found in every S. pyogenes strain investigated (Lukomski, et al., 2000; Rasmussen, Edén, & Björck, 2000). The scl1 gene is positively regulated by Mga (Almengor & McIver, 2004; Almengor, Walters, & McIver, 2006), and it is upregulated during biofilm formation and development (Cho & Caparon, 2005). Scl1 participates in S. pyogenes adherence to host epithelial cells and contributes to its virulence (Lukomski, et al., 2000). The role of Scl1 in the biofilm life cycle has also been evaluated.
Oliver-Kozup et al. demonstrated the importance of Scl1 to biofilm formation for a set of pathogenically diverse S. pyogenes strains (Oliver-Kozup, et al., 2011). Deletions of scl1 were created in MGAS6183 (M41), MGAS5005 (M1), MGAS6143 (M28), and static 24-hour biofilm assays in THY media on polystyrene were performed. Crystal violet analysis indicated that isogenic Δscl1 mutants had a substantially decreased average biofilm thickness, as compared to wild-type levels. Notably, MGAS315, a serotype M3 strain that possesses a naturally truncated scl1 allele, also failed to form a robust biofilm under the conditions tested. To further demonstrate the role of Scl1 in biofilm formation, Scl1 (and specifically the serotype M41 allele) was expressed in Lactococcus lactis MG1363. Expressing Scl1.41 on the surface was sufficient to confer increased biofilm formation capability on L. lactis when compared to the wild-type strain. Oliver-Kozup and colleagues concluded that Scl1 is a significant determinant for S. pyogenes biofilm formation (Oliver-Kozup, et al., 2011).
During the development of a biofilm, bacterial gene products are often up- or down-regulated in order to allow for the change from a planktonic to sessile state. For S. pyogenes, it has been demonstrated that the expression of a large number of specific genes are altered in this transition. Virulence factors such as the cysteine protease (SpeB), mitogenic factor (mf), and immunogenic secreted protein (isp) are upregulated in the biofilm state, as compared to the planktonic state. The virulence factor streptokinase (Ska) on the other hand, is downregulated in the biofilm state. It has been reported that genome wide, approximately 212 genes, or 14% are upregulated in the biofilm, and 203 genes or 13% are downregulated. Of these genes that display a changed gene expression profile, the majority of their roles involve one of the following categories: energy production and conversion, carbohydrate transport and metabolism, secondary metabolites biosynthesis, transport and catabolism, lipid transport and metabolism, and nucleotide transport and metabolism. This biofilm expression profile is altered further in the context of an in vivo infection. Further changes in gene expression have been observed in biofilms extracted from infected zebrafish muscle, which indicates that the host environment also plays a major role in the expression profile of biofilm-residing bacteria (Cho & Caparon, 2005).
The multiple gene activator of S. pyogenes, Mga, is responsible for the regulation of numerous virulence factors and regulates the expression of approximately 10% of the genome (Hause & McIver, 2012). Some of these virulence factors include M protein and M-like proteins (Hause & McIver, 2012). Mga has been shown to have a role in colonization and infection of the upper respiratory tract and deeper soft tissue in various animal models (Limbago, McIver, Penumalli, Weinrick, & Scott, 2001; Hondorp & McIver, 2007; Yung, McIver, Scott, & Hollingshead, 1999; Virtaneva, et al., 2005; Graham, et al., 2006). Mga may also play a role in biofilm formation, due to its role in autoaggregation (Cho & Caparon, 2005). The process of aggregation is thought to be one precursor to the formation of microcolonies and biofilms. Autoaggregation assays performed by Luo et al. reveal the importance of Mga in the initial stages of S. pyogenes biofilm formation. The WT Alab49 strain of S. pyogenes shows steady increases in autoaggregation, as demonstrated by a 75% decrease in turbidity over time. The mutant Δmga of the same strain shows low levels of autoaggregation, with a decrease in turbidity of only 5%. These data indicate that Mga-related genes are essential for autoaggregation and biofilm formation for strain Alab49 (Luo, Lizano, Banik, Zhang, & Bessen, 2008).
The transcription factor CodY can be found in S. pyogenes, as well as in other low G + C Gram positive bacteria. CodY is a global regulator that controls the transcription of a variety of genes, such as exoproteins, and is important in the response to amino acid depletion and nutritional stresses. When intracellular branched amino acids are plentiful, they bind to CodY, which results in an increased affinity for DNA binding and the subsequent repression of gene expression. When these amino acids are depleted, DNA affinity decreases, which allows for transcription. CodY has been shown to play a role in S. pyogenes biofilms under nutrient-depletion conditions. A ΔcodY mutant shows decreased static biofilms, as compared to its parent strain, when grown in CDM. No difference is observed in nutrient-rich media such as THY, which indicates that CodY has a minor effect on S. pyogenes biofilms under specific environmental conditions (McDowell, Callegari, Malke, & Chaussee, 2012).
CovR is a transcriptional regulator involved in the regulation of capsule production, as well as 15% of the S. pyogenes genome (Graham, et al., 2002). A covR mutant that contains capsule overexpression is unable to form a biofilm, as compared to its wild-type strain. This indicates that while capsule is known to be required for biofilm formation in certain strains, a CovR-regulated product other than capsule may also be involved in biofilm formation (Cho & Caparon, 2005).
Quorum sensing is the release of low molecular weight compounds between intra- and inter-species that results in a change in bacterial gene expression. This event depends on density and is used as a means of intercellular communication (Chang, LaSarre, Jimenez, Aggarwal, & Federle, 2011; Davies, et al., 1998), Currently, the S. pyogenes quorum sensing pathway is known to consist of the sil pathway, found in less than twenty percent of isolates (Belotserkovsky, et al., 2009; Eran, et al., 2007), and a highly conserved rgg-shp regulated pathway (Chang, LaSarre, Jimenez, Aggarwal, & Federle, 2011). Both of these pathways allow S. pyogenes to sense extracellular signaling peptides and respond to these cues by genome-wide changes in gene expression (Chang, LaSarre, Jimenez, Aggarwal, & Federle, 2011; Ibrahim, et al., 2007a; Fontaine, et al., 2010; Mashburn-Warren, Morrison, & Federle, 2010). Quorum sensing has been known to contribute to biofilm formation in several clinically relevant bacterial species, such as Pseudomonas aeruginosa (de Kievit, 2009) and Staphylococcus aureus (Kong, Vuong, & Otto, 2006), but its mechanisms in S. pyogenes have yet to be elucidated (Davies, et al., 1998).
The Rgg-SHP quorum sensing pathway is highly conserved among S. pyogenes strains (Chang, LaSarre, Jimenez, Aggarwal, & Federle, 2011). The Rgg-SHP pathway consists of the Rgg (regulator gene of glucosyltransferase (Sulavik, Tardif, & Clewell, 1992))-family of cytoplasmic receptors for intercellular signaling peptides in Streptococcal species (Ibrahim, et al., 2007a; Fontaine, et al., 2010; Mashburn-Warren, Morrison, & Federle, 2010). In S. pyogenes, four rgg-like genes have been identified: comR, ropB, rgg2, and rgg3 (Chang, LaSarre, Jimenez, Aggarwal, & Federle, 2011). Rgg2 and Rgg3 have antagonist activities, in which Rgg2 is an activator and Rgg3 is a repressor of gene expression (Chang, LaSarre, Jimenez, Aggarwal, & Federle, 2011). Additionally, rgg2 and rgg3 are adjacent to two small open reading frames that encode short hydrophobic peptides (SHPs) (Ibrahim, et al., 2007b). These two adjacent shp genes are important for positively regulating their own expression and inducing the expression of their neighboring genes, rgg2 and rgg3 (Chang, LaSarre, Jimenez, Aggarwal, & Federle, 2011). Furthermore, this Rgg-SHP pathway has been shown to be involved in S. pyogenes biofilm formation (Chang, LaSarre, Jimenez, Aggarwal, & Federle, 2011).
Chang et al. studied the Rgg-SHP system to determine its role in S. pyogenes biofilm development. Deletion strains were made in the wild-type NZ131, an M49 strain, for both rgg2 and rgg3. Crystal violet assays revealed the deletion of rgg3 had a three-fold increase in biofilm mass, as compared to the wild-type strain. The deletion of rgg2 resulted in a low production of biofilm, similar to the wild-type strain. ∆rgg2∆ rgg3 displayed low biofilm production, similar to that of the wild-type and the single deletion of rgg2 (Chang, LaSarre, Jimenez, Aggarwal, & Federle, 2011).
ropB is an rgg-like gene that encodes RopB, a positive regulator of speB transcription. SpeB is a S. pyogenes cysteine protease (Lyon, Gibson, & Caparon, 1998). Using the NZ131 parent strain, a deletion in the ropB gene was constructed to create a strain incapable of producing SpeB. The ∆ropB strain had enhanced biofilm formation, as compared to the wild-type strain. The double mutant ∆ropB∆rgg3 resulted in an additive effect on biofilm production. However, the double mutant ∆ropB∆rgg2 resulted in a decrease in biofilm formation, as compared to the single ∆ropB mutant, which eliminated the enhanced biofilm effects of the ropB deletion (Chang, LaSarre, Jimenez, Aggarwal, & Federle, 2011).
sSHP3-C8 is a synthetic peptide that contains the 8-C terminal amino acids of SHP3. The addition of exogenous sSHP3-C8 at a concentration of 50 nM caused a two-fold increase in biofilm production in the wild-type NZ131 strain. The addition of sSHP3-C8 to the ∆rgg3 mutant did not further increase its biofilm formation. In the ∆rgg2 and ∆oppD mutants (Opp: oligopeptide permease, D is a subunit of the transporter), biofilm formation was not stimulated upon sSHP3-C8 addition. When sSHP3-C8 was added to the ∆ropB mutant strain, there was a six-fold increase in biofilm formation, as compared to the wild-type. Chang et al. suggested that this observation can be attributed to the cells being more receptive to sSHP3-C8, that ∆ropB strain biofilms are more stabilized, or that the lack of SpeB protease in the ∆ropB strain allows for sSHP3-C8 to be more stable. Overall, Chang et al. suggest that there is a role for the Rgg2/3 pathway and its RopB counterpart in regulating the development of S. pyogenes biofilms (Chang, LaSarre, Jimenez, Aggarwal, & Federle, 2011).
Since shp genes can regulate their own expression and the expression of rgg2/3 genes, studies were performed to determine if surrounding genes contributed to the changes in biofilm formation. The proximal regions of the shp genes were deleted and these mutants revealed that proximal genes did not affect biofilm production. Therefore, Chang et al. suggest that genes near the rgg2/3-shp open reading frames are not likely to be involved in the biofilm process (Chang, LaSarre, Jimenez, Aggarwal, & Federle, 2011).
SilC (silC) is an important signaling peptide of the Sil (streptococcal invasion locus) quorum-sensing pathway in S. pyogenes (Hidalgo-Grass, et al., 2004; Eran, et al., 2007; Hidalgo-Grass, et al., 2002). It is known that silC is required for the virulence of the M14 strain JS95 during invasive soft-tissue infection (Hidalgo-Grass, et al., 2004; Hidalgo-Grass, et al., 2002). Additionally, silC has been investigated for its potential role in biofilm formation of strains from several different S. pyogenes serotypes (Lembke, et al., 2006; Thenmozhi, Balaji, Kumar, Rao, & Pandian, 2011).
Lembke et al. studied the potential role of the SilC signaling peptide in the biofilm development of S. pyogenes strains from serotypes M14 and M18. M14 and M18 silC-deficient mutants were assessed for their ability to form biofilms, relative to their respective wild-type strains. Strain M14 silC had been shown to have a reduced adherence to fibronectin, fibrinogen, and plastic, as compared to the parent strain; however, adherence was not statistically significant. An M18 silC-deficient mutant strain had significantly reduced adherence to collagen type I and IV substrates, as compared to its wild-type strain. The M14 silC-deficient mutant had a biofilm structure that was more cleft than its wild-type strain. The M18 silC-deficient mutant biofilm displayed a patchy, thin biofilm, as compared to the thick and solid wild-type biofilm (Lembke, et al., 2006).
Thenmozhi et al. screened a library of strains, including biofilm formers and non-biofilm formers, for the presence of the silC gene (signaling molecule). Thenmozhi et al. studied eleven S. pyogenes strains from different M serotypes (M49, M56, M63, M65, M74, M88.3, M89, M100, M122, st38, and st2147). Crystal violet staining was used to quantitate biofilms and categorize serotypes, according to those that were biofilm formers (M56, M65, M74, M89, M100 and st38), and those that were non-biofilm formers (M49, M63, M88.3, M122, and st2147). The silC gene was found in the genomes of some biofilm formers (M100, M74, and st38) and a single non-biofilm former (M122). The most proficient biofilm former observed in this study, M56, did not possess the silC gene. From their results, Thenmozhi et al. suggest that S. pyogenes strains from different serotypes have the ability to form biofilms, regardless of whether the silC gene is present (Thenmozhi, Balaji, Kumar, Rao, & Pandian, 2011).
Baldassarri et al. evaluated the ability of S. pyogenes clinical isolates to form biofilms, and investigated 289 clinical strains. The effect of temperature on biofilm formation was assessed for a set of 50 randomly chosen strains from the total of 289. Temperature appeared to have no effect on S. pyogenes growth, and so the growth conditions of 37°C and a THB medium were chosen for the remainder of the experiments. 18-hour biofilms were assessed by crystal violet to quantitate biofilms and were further examined by SEM to visualize the biofilm architecture. The number of strains capable of forming biofilms increased with the atmospheric conditions: 85% of isolates formed biofilms in unmodified atmosphere, 88% in 5% CO2, and 91.4% in anaerobiosis. Biofilm production did not vary for single isolates under the different atmospheric conditions. As a whole, there was a significant increase in biofilm formation under conditions of anaerobiosis when compared to conditions of 5% CO2 (Baldassarri, et al., 2006).
To study the effect of glucose on S. pyogenes biofilm formation, Thenmozhi et al. used a biofilm proficient strain, M100 (positive control), and a non-biofilm forming strain, st2147 (negative control). The results showed that st2147 can form biofilms with exogenous glucose supplementation. The addition of glucose (0.5–1.5%) was directly proportional to biofilm formation. Strain M100 produced biofilms profusely at 72 hours with the addition of all concentrations of glucose (0.5%, 1%, 1.5%). For both serotypes, a twofold increase in biofilm formation was observed when grown in media supplemented with 1.5% glucose (Thenmozhi, Balaji, Kumar, Rao, & Pandian, 2011).
The addition of glucose affected the surface area and thickness of the biofilms of both M100 and st2147 strains in different ways. Strain M100 had a decrease in surface area with increasing concentrations of glucose and reached its maximum surface area at 72 hours in both 0.5% and 1% glucose. An increase of glucose to 1.5% resulted in a significant elevation of the biofilm surface area of st2147 to 35% (Thenmozhi, Balaji, Kumar, Rao, & Pandian, 2011).
When the effect on biofilm thickness was evaluated, there appeared to be a significant decrease in thickness with increasing concentrations of glucose. The maximum biofilm thickness for strains M100 and st2147 was reached after 48 hours with varying concentrations of glucose. Strain M100 biofilms reached a maximum thickness of 14 microns in 1% glucose. The addition of 0.5% glucose resulted in a maximum biofilm thickness of 12 microns for strain st2147. Therefore, Thenmozhi et al. suggest that nutrients, such as glucose, play an important role in biofilm development (Thenmozhi, Balaji, Kumar, Rao, & Pandian, 2011).
The final stage in the biofilm life cycle involves single cells or groups of cells being released from an established biofilm. This cellular detachment facilitates biological dispersal, bacterial survival, and disease transmission. Dispersal can be achieved in either an active or passive manner. Active dispersal is initiated by the biofilm bacteria themselves, while passive dispersal involves external forces such as fluid shear, predatory grazing, interspecies antimicrobial compounds, and human intervention (Choi & Morgenroth, 2003; Erard, Miyasaki, & Wolinsky, 1989; Kaplan J. B., 2010; Lawrence, Scharf, Packroff, & Neu, 2002; Ymele-Leki & Ross, 2007).
At the cellular level, the methods by which cells release from a biofilm structure fall into three categories: erosion, sloughing, and seeding dispersal. Erosion is defined as the continuous release of single cells or small groups of cells at a low level throughout the course of biofilm development. Sloughing refers to an abrupt detachment of a large portion of biofilm, usually occurring during later stages of the biofilm life cycle (Lappin-Scott & Bass, 2001; Marshall, 1985; Stoodley, et al., 2001; Wilson, Hamilton, Hamilton, Schumann, & Stoodley, 2004). Seeding dispersal, also referred to as “central hollowing,” involves the rapid release of single cells or small groups of cells from hollow cavities formed inside the biofilm microcolony structure (Boles, Thoendel, & Singh, 2005; Ma, et al., 2009).
Although biofilm dispersal remains the least studied and understood aspect of biofilm development, a small number of mechanisms associated with biofilm dispersal have been identified. These include active biofilm dispersal, as well as chemical and environmental signals that regulate biofilm dispersal (Kaplan, 2010; Karatan & Watnick, 2009). Organisms can produce extracellular enzymes that degrade biofilm matrix components, as seen with dispersin B of Aggregatibacter actinomycetemcomitans (Kaplan, Ragunath, Ramasubbu, & Fine, 2003a) and the surface protein-releasing enzyme (SPRE) of Streptococcus mutans (Lee, Li, & Bowden, 1996). Enzymes produced by biofilm cells can also degrade biofilm substrates. Streptococcus intermedius hyaluronidase (Pecharki, Petersen, & Scheie, 2008) and the hemagglutinin protease of Vibrio cholerae (Finkelstein, Boesman-Finkelstein, Chang, & Häse, 1992) are two examples of such enzymes.
Seeding dispersal has been well characterized in A. actinomycetemcomitans (Kaplan, Ragunath, Ramasubbu, & Fine, 2003a; Kaplan, Meyenhofer, & Fine, 2003b; Kaplan & Fine, 2002) and Pseudomonas aeruginosa (Sauer, Camper, Ehrlich, Costerton, & Davies, 2002; Boles, Thoendel, & Singh, 2005; Ma, et al., 2009; Pamp & Tolker-Nielsen, 2007; Hunt, Werner, Huang, Hamilton, & Stewart, 2004; Schooling, Charaf, Allison, & Gilbert, 2004). Along with seeding dispersal, P. aeruginosa can also produce rhamnolipids, which are extracellular surfactants that decrease the adhesiveness of cell to surface interactions (Soberón-Chávez, Lépine, & Déziel, 2005; Neu, 1996), in order to facilitate its transition to a planktonic state. Other mechanisms include: modulation of fimbrial adherence, as in enteroaggregative Escherichia coli (EAEC) (Sheikh, et al., 2002; Velarde, et al., 2007) and enteropathogenic E. coli (EPEC) (Cleary, et al., 2004; Knutton, Shaw, Anantha, Donnenberg, & Zorgani, 1999); cell division-mediated dispersal (Allison, Evans, Brown, & Gilbert, 1990; Gilbert, Evans, & Brown, 1993); and induction of motility, as seen in E. coli and P. aeruginosa (Purevdorj-Gage, Costerton, & Stoodley, 2005; Jackson, et al., 2002).
Little is known about the ways in which S. pyogenes actively regulates biofilm dispersal. There is increasing evidence that the streptococcal regulator of virulence (Srv) and the streptococcal cysteine protease (SpeB) may play a major role in this process. Srv is a Crp/Fnr-like transcriptional regulator with homology to PrfA of Listeria monocytogenes (Reid, Montgomery, & Musser, 2004). The deletion of srv results in decreased biofilm formation, coupled with increased levels of SpeB production both in vitro and in two different animal models of infection (Doern, et al., 2009; Roberts A. L., Connolly, Doern, Holder, & Reid, 2010; Connolly, Roberts, Holder, & Reid, 2011a; Reid, et al., 2006; Roberts, Holder, & Reid, 2010a; Connolly, Braden, Holder, & Reid, 2011b). The addition of proteinase K is sufficient to inhibit biofilm formation and disrupt established biofilms in vitro (Doern, et al., 2009; Connolly, Braden, Holder, & Reid, 2011b), which indicates that S. pyogenes biofilm structures contain a protein component. Specific chemical inhibition of SpeB with the protease inhibitor E64 as well as allelic replacement of speB are sufficient to restore wild-type levels of biofilm formation in strains that lack Srv in vitro and in vivo (Doern, et al., 2009; Connolly, Braden, Holder, & Reid, 2011b). Western blot analysis and immunofluorescence microscopy can detect elevated levels of SpeB associated with strains that lack Srv (Doern, et al., 2009; Connolly, Roberts, Holder, & Reid, 2011a). Taken together, these data support a S. pyogenes biofilm regulation model whereby Srv acts as a repressor of SpeB, which maintains proteinase levels low enough to allow for biofilm formation (Figure 2). When the proper cues signal S. pyogenes to disperse from the biofilm, Srv repression is alleviated, SpeB levels increase, and subsequent degradation of protein components in the biofilm matrix allow biofilm cells to return to a planktonic state.
Due to the presence of DNA and protein in the matrix material of S. pyogenes biofilms (Doern, et al., 2009), it is also possible that an unidentified DNase or protease may be involved in the regulation of biofilm dispersal. Notably, the relationship between Srv and SpeB in relation to biofilm dispersal was shown across multiple serotypes (Connolly, Braden, Holder, & Reid, 2011b).
S. pyogenes biofilms provide protection against some antibiotics, but do not confer complete resistance to some antibiotics, such as penicillin (Baldassarri, et al., 2006; Conley, et al., 2003). Therefore, penicillin is typically the drug of choice for S. pyogenes infections, but can be replaced with macrolides (such as erythromycin) for patients with penicillin allergies. The increased use of macrolides in the treatment of S. pyogenes infections has resulted in increased resistance, according to the CDC.
Baldassarri et al. studied the relationship between macrolide resistance and biofilm formation among 289 S. pyogenes isolates. Using polymerase chain reaction (PCR) analysis, macrolide resistance genes were found in 122 strains. 50 strains contained erm(B), 36 strains possessed mef(A), 10 strains had erm(A) subclass erm(TR), and the other strains had a combination of these. Additionally, crystal violet staining used to quantitate the biofilms indicated that macrolide-susceptible S. pyogenes isolates produced significantly more biofilm than resistant strains. More specifically, isolates possessing genes that encode macrolide resistance (such as erm(B)- and erm(A) subclass erm(TR)- positive strains) through the methylation of 23S rRNA appeared to negatively affect biofilm production, as compared to susceptible strains and mef(A)-positive strains. The emm6 isolates, the strongest biofilm producers, and three of the strong biofilm-forming emm77 isolates were susceptible to macrolides. Of the other 15 emm77 isolates in this study, all but one contained one of the erm genes of macrolide resistance. There was no effect on biofilm production for those isolates that were tetracycline-resistant (Baldassarri, et al., 2006).
Additionally, Baldassarri et al. investigated the relationship between the presence of the prtF1 gene and biofilm formation. The prtF1 gene has been strongly related to erythromycin resistance (Facinelli, Spinaci, Magi, Giovanetti, & Varaldo, 2001). Of the 76 isolates investigated, 57 were prtF1-positive and 19 were prtF1-negative. There was a significant increase in biofilm formation of the prtF1-negative strains compared to the biofilm formation of the prtF1-positive strains. A negative association was also confirmed between prtF1 and macrolide resistance mediated by 23S rRNA methylation by grouping the isolates based on the presence of these genes (Baldassarri, et al., 2006). Baldassarri et al. suggested that macrolide-susceptible S. pyogenes strains may use biofilm to escape antimicrobial treatments and survive within the host (Baldassarri, et al., 2006).
Since they were interested in the relationship between macrolide resistance and biofilm formation, Thenmozhi et al. classified six of the eleven serotypes in this study as biofilm forming M serotypes (M56, M100, M74, M65, st38, M89). Of these six serotypes, M56 was resistant to erythromycin and carried both the erm(B) and mef(A) genes. The M56 serotype formed the thickest biofilms at about 8µ, as compared to the susceptible isolates of other M-serotype biofilm formers. Of the five serotypes classified as non-biofilm formers (M49, M63, M88.3, M122, st2147), only M49 was macrolide resistant. Thenmozhi et al. suggest that there is a negative association between having macrolide resistance determinants and having the ability to form biofilms (Thenmozhi, Balaji, Kumar, Rao, & Pandian, 2011).
S. pyogenes exhibits worldwide resistance to the fluoroquinolone family of antimicrobials (Jacobs, 2005). However, sub-lethal concentrations of fluoroquinolones can inhibit S. pyogenes biofilm formation in a concentration-dependent manner (Balaji, Thenmozhi, & Pandian, 2013). The metabolic activity of biofilm-residing S. pyogenes and biofilm biomass is also decreased after exposure to various derivatives of fluoroquinolone (Shafreen, Srinivasan, Manisankar, & Pandian, 2011).
There is still much to know about biofilm formation, regulation, dispersal, and its impact on S. pyogenes disease. The present evidence suggests that variation will continue to be present among strains, and points to the likelihood that the biofilm changes in nature to suit the environmental conditions and genetic repertoire of the strain in question. Regardless, further examination is likely to reveal regulatory pathways and integral components of biofilm structure that may serve as important therapeutic targets.
Except where otherwise noted, this work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License (CC-BY-NC-ND 4.0). To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-nd/4.0/
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