Bacterial Virulence Factors and Their Contribution to Pathophysiology after Thermal Injury

Invasion of host cell and immune system evasion

Bacteria use long hair-like projections called pili to attach to host cell surface receptors or extracellular matrix (ECM) proteins [5]. Bacteria can also move through transcellular spaces to get through epithelial surface of the host. Once inside the host cell, they can avoid uptake by phagosomes, actively destroy phagosomes with toxins, prevent phagolysosome formation, or exit phagosomes and survive in the intracellular milieu [5].

Quorum sensing and formation of biofilms

Bacteria use quorum sensing to communicate intercellularly via secretion of small molecules called autoinducers. When sufficient numbers of bacteria are present in an environment, autoinducer concentrations reach threshold leading to changes in production of VFs and formation of biofilms, which allow bacteria to share nutrients and resist antibiotic agents [5,6].

Biofilms are sessile communities of microbes that adhere to biotic and abiotic surfaces via an ECM composed of polysaccharides, proteins, and extracellular DNA. Biofilm formation depends on quorum sensing, and intracellular secondary messenger c-di-GMP system [7,8].

Biofilms form on catheters and ventilator tubes [9] and on chronic wounds with poor circulation and reduced immune response. Thus, large wounds and the presence of indwelling devices make burn patients especially vulnerable to infection by biofilm-forming pathogens [7]. The serum of burn-injured rats has been shown experimentally to induce oxidative stress that increases the biofilm formation of Staphylococcus aureus [10].

Biofilm ECM traps and prevents diffusion of antibodies, and prevents phagocytosis by neutrophils and macrophages that cannot effectively penetrate the ECM [9]. Pathogens within a biofilm also exhibit greatly altered expression of VF for example, Pseudomonas aeruginosa within a biofilm have been shown to increase production of rhamnolipids that degrade neutrophils [9], whereas Staphylococcus aureus express the leukocidins PVL and HlgAB [11]. Biofilms facilitate horizontal gene transfer between microbes, increase transfer of plasmids via conjugation [8,12], and promote mutations [13].

Endotoxins

The endotoxin lipopolysaccharide (LPS) is a major component of the outer cell membrane of gram-negative bacteria. Lipid A attaches LPS to the cell membrane and is recognized by toll-like receptor 4 (TLR-4) in human beings leading to production of proinflammatory cytokines resulting in endotoxic shock [14].

Superantigens

Superantigens are highly potent toxins produced by Staphylococcus aureus, Streptococcus, Mycoplasma, and Yersinia spp. Superantigens (SAgs) have the ability to crosslink major histocompatibility complex (MHC) class 2 molecules on antigen-presenting cells (APCs) and T-cells resulting in T-cell activation without the antigen processing and presentation [15,16]. As a result, there is an activation of exaggerated cytokine response [17] and in severe cases results in toxic shock syndrome.

Staphylococci

Staphylococcus spp. are the gram-positive bacteria most frequently identified in burn wound infections [3,18]. Staphylococcus aureus is part of the normal flora of human nares in up to 30% of the population [19]. It is a catalase- and coagulase-positive facultative aerobe responsible for cutaneous wound infections, pneumonia, endocarditis, osteomyelitis, toxic shock syndrome, and sepsis [20]. Coagulase-negative staphylococci such as Staphylococcus epidermidis are also known to infect burn wounds [3,21].

Staphylococcus aureus can produce approximately two dozen distinct cell wall-anchored surface proteins [22,23], that promote adhesion to host epithelium, ECM, and plasma proteins including fibrinogen, collagen, and fibronectin [23]. Staphylococcus aureus evade innate and adaptive immune responses through various mechanisms (Table 1). Protein A, a cell-surface protein encoded by the spa gene, prevents opsonization by binding directly to the Fc region of immunoglobulins, and when secreted may suppress B-cell activity via the cross-linking of B-cell surface proteins. Staphylococcus aureus additionally produce and secrete a range of VFs, including toxins, which inhibit neutrophil chemotaxis (CHIPS, FLIPr) and complement activation (SCIN), induce lysis of leukocytes and epithelial cells (pore-forming toxins, or PFTs), and interfere with adenosine signaling (AdsA) [24]. Staphylococcus aureus can both survive and reproduce within phagocytic immune cells such as neutrophils and macrophages and may even be transported throughout the host within these cells. They survive phagocytosis by production of enzymes such as catalase and superoxide dismutase that degrade reactive oxygen species during the oxidative burst, as well as enzymes that deacidify or damage the membrane of the phagosome [25].

The primary quorum-sensing gene cluster in Staphylococcus aureus is the accessory gene regulatory (agr) system. This system utilizes autoinducing peptides to regulate the production of numerous VFs via a positive-feedback mechanism. Furthermore, agr system is sensitive to abiotic and biotic environmental cues including pH, reactive oxygen species, and presence of blood serum proteins [26]. Inhibition of agr-mediated quorum sensing has been shown to reduce Staphylococcus aureus colonization and production of certain VFs in murine skin-wound infection models [27].

Staphylococcus aureus produces 19 different SAgs, including toxic shock syndrome toxin 1 (TSST1), staphylococcal enterotoxins (SE) A-R, and U [16]. Originally named enterotoxins because they were thought to only cause food poisoning, TSST1 was however found to be the cause of menstrual toxic shock syndrome, and burn wound sepsis [15]. Toxic shock syndrome toxin 1 has been isolated from serum and wounds of burn patients [28,29]. Prindeze et al. [28] assayed the serum of 207 adult burn patients and found exotoxins α-hemolysin, SEA, SEB, and TSST1 to be present in 45%, 25%, 13.5%, and 3.8% of patients, respectively. They concluded SAgs in burn patients' blood may contribute to morbidity and mortality [28].

Mino et al. [29] evaluated the presence of TSST1 and SEB in methicillin-resistant Staphylococcus aureus (MRSA) burn wound infections using a Sprague-Dawley rat model. They found that TSST1 was present on skin on all days with highest levels on day six, whereas SEB was found on skin and in the kidneys on days six and 10. This study concluded that bacteria in thermal wounds produce exotoxins that localize to end organs from skin in absence of bacteremia [29].

Panton-Valentine leucocidin (PVL) is an exotoxin produced by Staphylococcus aureus that along with α-hemolysin causes necrotizing pneumonia and soft tissue infections. Panton-Valentine leucocidin (PVL) is a pore-forming toxin that induces neutrophil lysis [30]. Its expression is not, however, the sole determinant of severity [31]. Khosravi et al. [32] found PVL to be present in 7.23% of MRSA and 33% of methicillin-susceptible Staphylococcus aureus (MSSA) strains isolated from burn wounds [32]. Panton-Valentine leucocidin may also be present in ventilator-associated pneumonia (VAP), seen in 27%–60% of burn patients [33].

Methicillin-resistant Staphylococcus aureus was first described in the 1960s and has since become a noteworthy nosocomial pathogen, accounting for at least 25%–50% of Staphylococcus aureus infections in hospitals [34]. A recent meta-analysis reported that burn patients in the intensive care unit (ICU) were 55% more likely to contract a MRSA infection than a MSSA one [35], and some burn units report MRSA infection rates higher than 50% [18]. The resistance of MRSA to methicillin, penicillins, and most other β-lactam antibiotic agents is conferred by the mecA gene, part of a mobile genetic element referred to as SCCmec. mecA is present in several staphylococcus species and encodes an alternative penicillin-binding protein (PBP2a) with a reduced affinity for β-lactams [34]. Vancomycin has been the most common treatment for MRSA infections, but recently, vancomycin-resistant Staphylococcus aureus (VRSA) and strains with intermediate resistance to vancomycin (VISA) have been described; new antimicrobial agents including linezolid, tigecycline, and daptomycin have been developed specifically to address vancomycin-resistant strains of Staphylococcus aureus [36].

Enterococci

Enterococcus spp. are the second most common gram-positive pathogens isolated from burn wounds. Enterococcus faecalis and Enterococcus faecium are versatile and abundant bacteria found in soil, seawater, and human gut [37]. Vancomycin-resistant enterococcus (VRE) have recently emerged as a clinical cause of infection [18,37].

Enterococcus spp. express several microbial surface components recognizing adhesive matrix molecules (MSCRAMMs) that contribute to virulence. Enterococcus faecalis, expresses the collagen-binding protein Ace and the adhesive pili ebp during infection and promote colonization of the host [37]. The pilus protein ebp and the enterococcal polysaccharide antigen (Epa) are ubiquitous in clinical Enterococcus faecalis strains and contribute to virulence and biofilm formation [38]. Clinical Enterococcus faecium strains also express collagen-binding proteins including Acm [37,39] and adhesive pili [40]. SagA is a protein secreted by Enterococcus faecium that has a high affinity for ECM proteins and promotes both colonization and biofilm formation [41]. Several putative MSCRAMM and pili genes have been identified in Enterococcus spp. that are not well characterized [40,42]. Enterococcus spp. additionally produce and secrete cytolysins encoded by cylL genes and gelatinase (GelE), a metalloproteinase that degrades gelatin and collagen. Quorum-sensing in Enterococcus faecalis is primarily mediated by the fsr locus and the cytolysin operon, which control biofilm formation and production of proteases and cytolysins (Ali). The quorum-sensing mechanisms of Enterococcus faecium are not well-characterized [37].

Enterococcus spp. is intrinsically tolerant to β-lactams because of pbp5, a gene that encodes a penicillin-binding protein with low affinity for antibiotic agents including ampicillin and cephalosporins [43]. A sequence variation in Enterococcus faecium pbp5 known as Pbp5-R confers high-level ampicillin resistance and is common in hospital-associated strains of Enterococcus faecium. The blaZ gene, encoding a secreted β-lactamase, may also be present in Enterococcus spp. And contributes to ampicillin resistance. At least nine van gene clusters (vanA-vanE, vanG, vanL, vanM, vanN), conferring vancomycin resistance, have been identified in enterococci, with vanA and vanB being the most common in clinical isolates [37,43]. More than 80% of clinical Enterococcus faecium strains, and 5% of Enterococcus faecalis strains are resistant to vancomycin [38]. Enterococci may also acquire high-level resistance to aminoglycosides via mutations in rRNA sequences or genes encoding aminoglycoside-modifying enzymes that are spread on mobile genetic elements [37,38]. Mutations in the gyrA and parC genes of enterococci have been observed to confer fluoroquinolone resistance. Although rare, resistance to linezolid and daptomycin may be acquired through gene mutations [38].

Streptococci

Streptococcus spp. were once a common source of burn wound infection and mortality, but the penicillin G susceptibility of these species have made them less clinically relevant in the past few decades [3]. Briefly, Streptococcus pyogenes contains surface proteins called M proteins, that along with polysaccharide capsule, help the pathogen evade phagocytosis by the host immune system. Additionally, they secrete hemolytic toxins, and streptolysins that break down cell membranes of various host cells [44]. Streptococcus pyogenes superantigens are exotoxin A and C (SPEA, SPEC) and streptococcal mitogenic exotoxin Z (SMEZ) [14]. Streptococcus pyogenes strains are almost universally susceptible to β-lactams, but they may gain some tolerance if they grow together with a β-lactamase–producing bacterium such as Staphylococcus aureus [45]. Resistance to aminoglycosides is rare in streptococci. The susceptibility of these bacteria to penicillin and aminoglycosides make them less clinically important in burn patients [45].

Pseudomonas

Pseudomonas aeruginosa is a gram-negative facultative anaerobe found widely in water and soil. It is an opportunistic pathogen, commonly causing airway, urinary tract, and wound infections, particularly in immunocompromised patients [46–48]. Its preference for damp environments causes it to grow in burn wounds. Furthermore, it is a common cause of sepsis in burn patients [18] and the most common cause of nosocomial infections [49].

Pseudomonas aeruginosa attach to the host cells in the airway, on the skin and in chronic wounds. Pili also aid in movement of bacteria on host cells and promote biofilm formation. Lipopolysaccharide, an endotoxin present on the cell surface of Pseudomonas aeruginosa also promotes adherence to the host cell and is responsible for eliciting inflammatory response in the host [48].

The mechanisms of Pseudomonas aeruginosa immune evasion are not well understood and are thought to be tightly regulated by quorum-sensing and other intercellular signaling pathways (Table 2) [50,51]. The type 3 secretion system, a needle-like structure produced by Pseudomonas aeruginosa, can inject immune cells with effector toxins including exoenzymes S, T, U, and Y that allow it to escape host defenses. Generally, either exoS or U gene will be present in one bacterial strain. ExoS, T, and Y disrupt cell junctions in addition to preventing phagocytosis. ExoU utilizes phospholipase A2 activity to break down host phospholipid cell membranes, leading to cell lysis [52]. Furthermore, it activates cyclo-oxygenase-mediated inflammatory pathways [48,53]. Presence of the type 3 secretion system is implicated in severe lung infections [52].

Pseudomonas aeruginosa secretes exotoxin A that binds to the α2 macroglobulin receptor on fibroblasts and enters the host cell endoplasmic reticulum (ER) and subsequently the cytosol. There, it ADP-ribosylates and thus inactivates elongation factor-2 that stops protein synthesis, leading to apoptosis [54,55].

Expression of VFs including pyocyanin, alkaline protease, and exotoxin A are heavily influenced by Pseudomonas aeruginosa autoinducer concentrations [47]. Mutation of the quorum-sensing genes las1 and rhl1 has been shown to reduce virulence and mortality in a murine burn-wound infection model [56]. Inhibition of quorum-sensing has also been observed to mitigate the increased intestinal permeability associated with Pseudomonas aeruginosa infection in a mouse burn model [57].

Several VFs that confer antimicrobial resistance can be upregulated in Pseudomonas aeruginosa within a biofilm. These include β-lactamases that are secreted into the ECM, the NdvB glucosyltransferase that produces cyclic glucans that, in the ECM, neutralize aminoglycosides including kanamycin and tobramycin, and the BrlR locus, a transcriptional activator for multi-drug efflux pump genes [8,9].

A study of 144 Pseudomonas aeruginosa isolates collected from infected burn wounds in Tehran, Iran, found that 92.4% produced biofilms, and that multi-drug–resistant strains were more likely to produce biofilm [58]. In a rat scald-wound model, full and partial-thickness burn wounds inoculated with Pseudomonas aeruginosa showed substantial development of biofilms that had increased expression of various VF compared with the planktonic inoculum [59].

Pseudomonas aeruginosa has intrinsic resistance to a variety of antibiotic agents [49]. It is attributed to its low number of non-specific porins and its expression of several multi-drug efflux pump genes including MexAB-OprM, MexXY/OprM, MexCD-OprJ, and MexEF-OprN, the expression of which are known to be linked to the quorum-sensing las system [47]. Pseudomonas aeruginosa also possess a gene, ampC, encoding a β-lactamase, which confers resistance to most β-lactams and is highly expressed by the majority of clinical Pseudomonas aeruginosa strains [47,49]. Pseudomonas aeruginosa may acquire specific or non-specific antimicrobial resistance by mutations that confer resistance to β-lactams, carbapenems, and aminoglycosides. Furthermore, it can increase production of efflux pumps and ampC by altering regulatory machinery via gene mutations. This leads to enhanced resistance to β-lactam antibiotic agents [47,49,60].

Acinetobacter baumannii

Acinetobacter baumannii is a rod-shaped, non-spore forming opportunistic bacterium that causes nosocomial infections in burn patients in the ICU [61]. It has been found in respiratory samples from combat-injured patients [62]. It is naturally found in soil and water and has been isolated from ventilators, humidifiers, and other surfaces in the hospital. Acinetobacter is part of the normal skin flora in approximately 40% of the population and can colonize the respiratory tract. It is an emerging cause of VAP and indwelling catheter-associated infections [63] and leads to increased length of hospital stay [64]. It is four times more likely to develop drug resistance than Pseudomonas and Klebsiella [63].

Acinetobacter baumannii VFs aid in motility, biofilm formation, and antimicrobial resistance. The Acinetobacter baumannii capsule consists of repeating units of polysaccharides that allow it to survive desiccation by disinfectants. It can also enter a dormant state in the absence of water. The RecA protein, involved in homologous recombination, prevents DNA damage in the dehydration-rehydration cycle of Acinetobacter baumannii. It may evade damage from hydrogen peroxide by increasing synthesis of proteins involved in detoxifying reactive oxygen species. Furthermore, an efflux system pumps chlorhexidine out of Acinetobacter baumannii cells. It is also able to evade phagocytosis in the presence of ethanol, and thus causes infections in chronic alcohol abusers [63].

The polysaccharide capsule is one of the most virulent molecules of Acinetobacter that helps evade the host complement system. Strains lacking a capsule have been found to be avirulent. Because of the variability in polysaccharide composition between strains, it has been difficult to develop a vaccine against the capsule.

Acinetobacter baumannii makes biofilms on cutaneous wounds, dressings and medical equipment via a type 1 chaperon-usher (Csu) pilus system. Furthermore, biofilm-associated proteins (BapAb) are responsible for maintenance of biofilms on titanium and polystyrene. Other molecules that aid in biofilm production are poly-β-(1-6)-N-acetylglucosamine (common to gram-negative bacteria), the capsule, and the autotransporter system. Acinetobacter baumannii outer membrane protein A (OmpA) also helps in attachment to the epithelial cells and form biofilms. By disrupting OmpA, a murine model has shown to have reduced mortality [65].

The outer cell membrane of Acinetobacter baumannii does not contain O antigen and is called lipooligosaccharide. It functions in a similar manner to the LPS found in other gram-negative bacteria and stimulates synthesis of interleukin (IL)-8 and tumor necrosis factor (TNF) in infected humans via a TLR-4–mediated pathway. It can also help the pathogen develop antibiotic resistance. Furthermore, resistance to colistins is developed by altering the structure of lipid A and preventing binding of the antibiotic.

Acinetobacter baumannii uses metal ions from the host for its metabolic needs. It has developed various siderophores, iron chelating molecules, that bind iron especially in the acidic environment of an infection. Zinc is another important cofactor and humans sequester it bound to calprotectin. One-way Acinetobacter baumannii deals with zinc deficiency is via a high-affinity zinc acquisition system (ZnuABC99). It is a transcription repressor that under normal conditions blocks transcription of genes regulated by zinc. During conditions of zinc deficiency, this block is lifted, and gene transcription occurs in absence of zinc [63,65].

Enterobacteriaceae

Several members of the Enterobacteriaceae family including Escherichia coli, Klebsiella pneumoniae, Enterobacter spp., and Proteus spp. cause respiratory and urinary tract infections (UTIs) in burn patients [3,18]. As with other bacterial species, these bacteria are developing resistance to antibiotic agents, especially to carbapenems [18,66].

Klebsiella pneumoniae

Klebsiella pneumoniae is a rod-shaped, lactose-fermenting gram-negative opportunistic pathogen that is part of the normal flora of the mouth, skin, and gastrointestinal tract [67]. It causes wound infections [68,69], pneumonia and UTIs in burn patients [18].

The Klebsiella pneumoniae capsule helps the pathogen evade phagocytosis by host immune cells. Highly virulent strains have increased expression of the capsule polysaccharide (CPS), which inhibits TLR-2 and TLR-4–mediated inflammatory pathways. Furthermore, secreted CPS binds to antimicrobial molecules released by the host cell thus preventing them from attaching to bacteria. Capsule polysaccharide can also affect dendritic cell maturation and inhibit production of TNF-α and IL-12. The O-antigen of Klebsiella pneumoniae LPS prevents complement-mediated killing, whereas lipid A prevents phagocytosis. Along with CPS, O-antigen allows bacteria to travel in the blood, leading to sepsis. Capsule polysaccharide alone causes pneumonia by complement molecule C3 deposition and preventing phagocytosis by alveolar macrophages. Klebsiella pneumoniae type 1 fimbriae aids in bacterial attachment to mannose structures on host cells and contributes to the development of UTIs. Type 3 fimbriae play a role in biofilm formation in the setting of urinary catheters. Kpc fimbriae is also involved in biofilm formation and KPF-28 aids in intestinal colonization via binding to Caco-2 cell lines. Similar to other gram-negative bacteria, Klebsiella pneumoniae contains outer membrane protein A (OmpA) that prevents activation of airway epithelial inflammatory response. Efflux pumps allow transport of host antimicrobial peptides and antibiotic agents out of bacterial cells, thus allowing them to evade host immune systems and develop antibiotic resistance, respectively. Klebsiella pneumoniae involved in respiratory tract infection have developed siderophores for iron acqusition. Furthermore, Klebsiella pneumoniae strains that live in the host gastrointestinal tract, contain urease, and undergo urea metabolism [67].

Proteus mirabilis

Proteus is a gram-negative bacillus that normally resides in soil, water and gastrointestinal tracts. Proteus mirabilis is an opportunistic pathogen that causes infection of the respiratory tract, ear, nose, throat, eyes, and skin [70]. It routinely causes catheter-associated urinary tract infections (CAUTIs) and urinary stone formation. It also forms biofilms on catheters, leading to obstruction, and even replaced catheters may be colonized by pathogens present in urinary stones. Biofilms and stones consist of either struvite (magnesium, ammonium, phosphate) or apatite (calcium phosphate) crystals that give the pathogen an adequate environment for growth. One of the most important VFs of Proteus mirabilis is urease, which converts urea to ammonia and carbon dioxide, thus alkalinizing urine, leading to the precipitation of solid crystals found in biofilms and stones. Mutated and urease-negative Proteus mirabilis are unable to form stones [71].

The second VF involved in CAUTIs is mannose-resistant proteus-like (MR/P) fimbriae. These are a type of chaperone-usher, a common fimbria of gram-negative bacteria. The exact MR/P receptors are not known but it is theorized that MR/P plays a role in the adherence of Proteus mirabilis to the bladder epithelium [72].

Proteus mirabilis use flagella for a specialized form of movement called swarming, which is a coordinated movement of the whole colony. It not only moves the pathogens through solid and liquid phases but also helps seed new colonies from a pre-existing biofilm. Proteus mirabilis also contains two types of flagella that aid in motility and evade the host immune system, promoting infection. There is conflicting evidence in the literature about the role of Proteus mirabilis motility in infections because non-motile organisms have also been shown to cause UTIs. Proteus mirabilis also uses its swarming ability to transfer other pathogens such as Staphylococcus spp. on the catheter surface. Furthermore, Proteus mirabilis contains polysaccharide LPS, proteases, and iron acquisition mechanisms that are involved in infections [73].

Proteus mirabilis can form biofilms on all the urinary catheters currently in use. Studies have shown reduction of biofilms with triclosan-coated catheters and urinary stents, however, there is a risk of selection of resistant strains. Nalidixic acid and ethylenediaminetetraacetic acid (EDTA)-treated silicone catheters have shown some success in decreasing biofilm formation. Other compounds that have been tested with some success in laboratory settings are urease and quorum-sensing inhibitors, hydrogel, and silver coatings. However, these are not yet clinically available [70].

Escherichia coli

Escherichia coli is a gram-negative facultative anaerobic rod that is part of the normal gut flora. Extra-intestinal Escherichia coli strains can cause UTIs, neonatal meningitis, wound infections, and sepsis [74]. A study done by Yali et al. [75] collected blood and wound samples from 63 burn patients in the ICU and found Escherichia coli to be present in 8.6% of samples. Because of widespread use of antibiotic agents, certain strains of Escherichia coli have developed antibiotic resistance and may now lead to severe infections [74].

Escherichia coli produce several virulence factors including the capsule, fimbriae, toxins, proteases, and siderophores. The Escherichia coli polysaccharide capsule prevents phagocytosis by host cells and helps the pathogen escape complement molecules in the blood. It also prevents desiccation and aids in biofilm production [76]. Escherichia coli LPS initiates an inflammatory response in sepsis and contributes to antibiotic resistance. Plasmid ColV contains genes necessary for serum resistance and iron uptake in strains causing sepsis, including a gene encoding the siderophore aerobactin, which promotes iron scavenging. Other iron acquisition systems are yersiniabactin, ChuA, sitABCD system [74,77].

Escherichia coli surface fibers termed curli fibers are involved in cell communication during biofilm formation [78]. These bind to host cell laminin, fibronectin, plasminogen, and major histocompatibility complex class I [74]. Type 1 pili expressed by virulent Escherichia coli contain adhesins and contribute to attachment to host cells and biofilm formation [78]. Both of these molecules are potential targets for antimicrobials. Furthermore, fimbriae promote adherence to host cell membranes and abiotic surfaces, and outer membrane proteins contribute to biofilm formation [74,78]. Similar to other gram-negative bacteria, Escherichia coli utilize a type 3 secretion system to inject bacterial protein into mammalian cells [74]. Antibiotic resistance to Escherichia coli is conferred by β-lactamases, extended spectrum β-lactamases, and carbapenamases [79].