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Incidences of antimicrobial resistant infections have increased dramatically over the past several decades and are associated with adverse patient outcomes. Alternative approaches to combat infection are critical, and have led to the development of more specific drugs targeted at particular bacterial virulence systems or essential regulatory pathways. The purpose of this review is to highlight the recent developments in anti-bacterial therapy and the novel approaches toward increasing our therapeutic armory against bacterial infection.
Although classic antibiotic development is not occurring rapidly, alternative therapeutics that target specific bacterial virulence systems are progressing from the discovery stage through the FDA approval process. Here we review novel antibodies that target specific virulence systems as well as a variety of newly discovered small molecules that block bacterial attachment, communication systems (quorum sensing) or important regulatory processes associated with virulence gene expression.
The success of novel therapeutics could significantly change clinical practice. Furthermore, the complications of collateral damage due to antibiotic administration e.g. suprainfections or decreased host immunity due to loss of synergistic bacterial communities, may be minimized using therapeutics that specifically target pathogenic behavior.
The rapid increase in bacterial antimicrobial resistance remains a growing problem, decreasing the therapeutic options available for a multitude of pathogenic species that commonly infect the human host. The purpose of this review article is to examine the developing field of alternative therapeutics that combat bacterial virulence systems of specific pathogens associated with poor patient outcomes. This review also examines evidence for polymicrobial communities in a variety of clinical infectious states and discusses the utility of combined therapeutic interventions to improve long-term patient outcome.
Our long term relationship with bacteria initiates during the birthing process and culminates in a human super-organism that contains 10 times more microbial than human cells . Disturbances in this delicate balancing act, shift the host-microbe interaction from one of synergy to infection, resulting in host tissue damage and the need for therapeutic intervention. Since the relatively recent discovery of Penicillin as the active antimicrobial agent produced by Penicillium species, a multitude of antibiotics discovered and chemically modified over the past 8 decades have provided the first line of defense against a variety of bacterial infections. Historically, these compounds have dramatically changed our ability to combat infectious agents. However, bacteria are adept at rapidly acclimatizing and developing resistance to a myriad of insults, including antimicrobial agents which they commonly encounter in non-host environments, resulting in more resistant pathogens and increasingly limited treatment options.
Since antibiotic compounds are currently being developed at a significantly slower pace than the evolution of bacterial resistance, alternative strategies are necessary to combat infecting species. Approaches vary from targeting specific virulence systems to manipulating physiological aspects of bacterial lifestyle such as cell-to-cell communication or the need for a specific factor for virulence gene expression. The following, while by no means a comprehensive discussion, provides an overview of some of the current approaches being used to develop alternative antimicrobial therapies to combat bacterial infection in the human host.
One approach to develop novel antimicrobial compounds is to target specific virulence factors associated with a particular disease state. Secretion systems and toxins provide ideal targets for novel therapies and many studies have focused on developing treatments to neutralize or block these specific physiological factors.
The type III secretion system (TTSS) found in many pathogenic species is a needle like injection system that facilitates secretion of effector molecules directly into the mammalian host epithelium (Fig. 1A). This specific virulence system in clinical isolates of Pseudomonas aeruginosa has been strongly correlated with acute lung injury and the development of ventilator-associated pneumonia in intubated patients [2–5]. Active type III cytotoxin secretion also targets host immune cells e.g. macrophage and neutrophils, allowing organisms evade the immune response and often reducing the numbers of viable host defense cells [6, 7].
The type III injectosome is comprised of multiple proteins which anchor the needle complex in the bacterial membrane and facilitate toxin secretion. For example, the PcrV protein facilitates P. aeruginosa cytotoxin secretion and is located at the tip of the needle apparatus . This protein is believed to play a role in binding mammalian membrane receptors (Fig. 1B) and is essential for effective cytotoxin secretion by this species. A novel antibody (anti-PcrV) which binds this protein exhibits excellent efficacy in protecting both acutely infected and burned mice against type III-mediated epithelial damage [9–13], presumably by blocking binding of the needle complex to the mammalian cell membrane preventing effector translocation (Fig. 1C). This antibody has been humanized to increase its half-life and reduce immunogenicity and is currently in human phase I/II clinical trials. Use of this antibody could conceivably provide a three-fold mechanism of protection by 1. preventing acute lung injury thus 2. reducing the inflammatory response which can also contribute to host epithelial injury and 3. preserving macrophage and neutrophil activity. A viable host defense system with greater numbers of functional immune cells would permit enhanced clearance of co-habiting species, particularly pathogenic ones that through their combined activity contribute to the disease state.
A similar approach has been used to develop antibodies against type III secretion by Yersinia pestis the causative agent of bubonic plague. LcrV, a protective antigen and the Y. pestis ortholog of PcrV, is also located at the tip of the needle-like type III injection apparatus. Like PcrV, LcrV facilitates secretion of the cytotoxic Yersinia protein effectors (Yops) [14, 15]. An antibody against this protein has demonstrated protection against lethal doses of a virulent Y. pestis strain in a mouse model of bubonic plague preventing host cell apoptosis and promoting phagocytosis 
Other antibody-based therapeutics in development include Valortim, a human monoclonal antibody which exhibits prophylactic and post-infection efficacy against B. anthracis toxins in rabbit and non-human primate models . This antibody has successfully completed Phase I safety and pharmacokinetic evaluation and has been fast-tracked by the FDA to facilitate rapid regulatory review. The anti-virulence antibody, “BAT” (heptavalent botulism antitoxin) has been developed to provide protection against 7 botulinum toxins (A, B, C, D, E, F and G). This improved antibody whose efficacy has been assessed in animal studies, currently has “investigational drug” (IND) status from the FDA for studies in human subjects (http://www.cnw.ca/fr/releases/archive/September2007/27/c6071.html).
Part of the typical bacterial lifecycle involves attachment to a surface and biofilm formation. Biofilms are broadly defined as communities of bacterial cells encased in a protective extracellular matrix. Due to the inherent increased resistance of biofilms to conventional antimicrobial therapy, clearance of these bacterial reservoirs is problematic, leading to subsequent infections. In the human host, this mode of bacterial lifestyle contributes substantially to chronic infections, such as airway colonization of cystic fibrosis patients [18–21] and recurrent urinary tract infections[22, 23]. The initial stages of biofilm formation involve attachment of the bacterial cell to the host cell surface, which is mediated by adhesion proteins. Blocking this initial attachment step provides a strategy for preventing colonization of the epithelial surface. Unfortunately, a multitude of adhesin proteins are produced by various bacterial species, thus, development of a common anti-adhesion therapy that would universally block bacterial attachment to host cells is unlikely. However, some pathogenic species such as P. aeruginosa and Candida albicans do appear to possess adhesions that have homologous receptor-binding domains. This aspect has been exploited to develop therapeutics effective against both species in parallel . Other anti-adhesion therapies that target species-specific adhesions e.g. those of uropathogenic Escherichia coli, the primary cause of recurrent urinary tract infection, are also currently in development.
Pili or fimbrae, hollow extracellular bacterial appendages composed of oligomreic pilin proteins, also play a prominent role in initial bacterial attachment, by binding receptors on the mammalian host cell surface [26–29]. Both Gram positive and Gram negative bacterial species produce pili making them an attractive target for novel therapeutics. Adhesins such as lectin are commonly found on the pili and facilitate binding to oligosaccharides on the mammalian cell surface . Approaches to prevent initial attachment events involving pili include blocking binding by the pili-associated adhesions. Naturally occurring and synthetic carbohydrate derivatives have exhibited effective pili-blocking activity in a variety of assays [31–33], and represent a viable option for novel anti-adhesive therapeutics . An alternative approach is to inhibit pilus formation by blocking the activity of chaperone-usher systems necessary for their biogenesis. Pilicides are in development for a number of bacterial species including Escherichia coli [35–38]. Given the key role pili play in initial adhesion and the fact that the chaperone-usher system plays a pivitol role in pili biogenesis for a number of pathogenic species, it is hoped that pilicides may be effective against multiple species, providing a broad-range antibacterial therapeutic option.
A recent study to examine the molecular basis of how commensal species become pathogenic, demonstrated inhibition of Shigella species virulence gene expression by quinolinate. This compound produced by the nadA and nadB genes of Shigella species acts as an anti-virulence factor that inhibits invasion by blocking type III secretion of two effector proteins IpAB and IpaC that are key to mammalian cell invasion. In addition, quinolinate also prevented cell-to-cell spread of S. flexneri 5a and its ability to induce polymorphonuclear neutrophil transepithelial migration .
Screening libraries of natural products, small synthetic molecules and peptides is proving to be a fertile area for novel antimicrobials. To date, small molecule library screening has led to the identification of a compound, virstatin, which effectively inhibits expression of the toxT gene, a transcription factor that activates expression of cholera toxin and pilus production by Vibrio cholerae . In separate studies, compounds (peptides and small molecules) that block anthrax toxin by inhibiting the enzymatic activity associated with activity have been identified [41–43] while others (Amiodarone and Bepridil) block endosomal acidification – a crucial step in toxin entry to the mammalian cell . In another study, Nordfelth et al screened a library of small molecules to identify a compound that would inhibit Type III secretion by Yersinia pseudotuberculosis. This approach led to the identification of an acylated hydrazone that specifically blocks TTSS effector protein secretion by this species .
Bacteria are masters of primordial sensory perception; this ability is facilitated through a communication system called quorum sensing (QS) that permits individual cells of a single species to sense either “self” (cell density of same species) or “non-self” (distinct microbial neighbors). The complex hierarchical genetic network that underlies this signaling system provides exquisite regulatory control of specific subsets of genes, including virulence factors, permitting defined responses to changing environmental conditions . QS by both Gram negative and Gram positive bacterial species share the same principles, production of and sensing molecular signaling compounds. However, the mechanism by which these molecules are produced and sensed and the signal molecules themselves vary across species. QS has been associated with biofilm formation [47–49] and with regulation of the virulence factors associated with this sessile lifestyle .
In 1993, de Nys et al  reported that a marine alga, Delisea pulchra, produced a variety of halogenated furanones that inhibit bacterial quorum sensing and provided antifouling protection. This was the first report of quorum sensing inhibitors (QSI’s). Efforts to chemically modify these compounds to increase their activity against clinically relevant species such as P. aeruginosa resulted in identification of two compounds C-56 and C-30 which have demonstrated efficacy in clearance of this species in a mouse model of chronic infection [51, 52]. Other QSI’s have since been identified in herbal and fungal foods [53, 54], these compounds also demonstrate efficacy in vitro and in vivo in a murine model of infection. Recently, a QSI molecule, hamamelitannin, that occurs as a natural product in the bark of witch hazel was discovered by structure-based “virtual screening” . This compound demonstrated QSI activity and was shown to inhibit biofilm formation on in-dwelling devices in a rat model. What was particularly exciting about this compound was that it prevented infection by Methicillin Resistant Staphylococcus aureus (MRSA) and Methicillin resistant Staphylococcus epidermindis (MRSE) – two of the most problematic and intractable clinically relevant pathogens **.
To date, no study has examined the effect of QSI compounds on reciprocally regulated virulence factors that may be induced due to blocking QS-controlled biofilm-associated pathogenicity e.g. type III secretion in P. aeruginosa  or their effects on host polymicrobial community diversity or function. While these compounds hold promise, caution must be taken against extrapolating single species results with multi-species clinical reality. Inter-species cross-talk has been demonstrated between P. aeruginosa and Burkholderia cepacia  and between Serratia liquefaciens and P. aeruginosa . Molecules that block or potentially mimic these inter-species communication molecules may provide a means to manipulate the physiology of multiple species in parallel and another avenue for therapeutic development.
Another promising therapeutic approach aimed at the clinically relevant pathogen P. aeruginosa, targets a specific facet of this organisms physiology essential for its pathogenicity. The human immune response limits iron (Fe3+) availability in an attempt to attenuate virulence of infecting pathogens. However, Fe3+ plays a key role in many clinically-relevant pathogenic species and a variety of systems to scavenge Fe3+ from the environment have developed in these organisms. Recent work by a group in Seattle used a “Trojan Horse” strategy to deceive the bacterial cell and reduce iron uptake . The transition metal gallium (Ga3+) is chemically similar to iron, but unlike Fe3+ it does not under go redox reactions and therefore is biologically inactive and unable to perform the cellular functions of Fe3+ within the bacterial cell. Provision of gallium to established biofilms of P. aeruginosa in vitro resulted in a dose dependent clearance of these sessile communities . This approach has also demonstrated effectiveness in vivo in two murine models of infection . Presence of gallium reduces Fe3+ uptake therefore repressing iron-dependent transcriptional regulators such as pvdS that control a number of virulence factors in P. aeruginosa. Two benefits of using this approach in human hosts are that gallium is already approved for use in large concentrations by the FDA and since Fe3+ is required by a multitude of clinically relevant pathogens it is likely that this therapeutic will be efficacious against co-infecting pathogens in polymicrobial communities.
One key issue with development of species-specific therapies is that bacterial species do not exist in isolation in the human host. The evidence that co-habiting bacteria or polymicrobial communities occupy specific host niches has increased rapidly over the past 5 years. Prior to this, our perception of infection was limited to a handful of primary pathogens occurring in isolation in these niches. The advent of applying culture-independent approaches to examine clinical samples has revealed a wealth of previously unappreciated microbial diversity. For example, a recent survey of pediatric cystic fibrosis airways revealed that 65 different bacterial species could be identified and coincidence of known respiratory pathogens was consistently seen across patient samples . Recent culture-independent analyses of gastrointestinal microbial communities demonstrated the presence of 395 bacterial phylotypes, 62% of which represented novel lineages and that shifts in bacterial community composition underlie disease states [61–64]. Another study has demonstrated pathogen co-incidence in airway polymicrobial communities of patients developing ventilator-associated pneumonia . These and many other studies have demonstrated the complexity of our relationship with microbial inhabitants and underscores the likelihood that pathogenesis is mediated by a mixed population rather than a single species in isolation.
While the usual pathogenic suspects remain key species in these bacterial communities, our perception of infection is changing from one of a single species phenomenon to that of a dynamic polymicrobial state composed of multiple co-incident pathogens. Bacteria possess a multitude of two-component signal transduction systems that allow them to sense environmental signals and respond appropriately. In infectious states, these signals may come from the host immune response or the microbial neighbors co-habiting the same ecological niche. These insights now allow us to consider therapeutic approaches that block specific virulence factors or dull the sensor-response systems of the resistant species within the polymicrobial community preventing host cell damage.
Culture-independent tools are permitting greater insight into how interventions such as antimicrobial administration cause substantial collateral damage to microbial diversity, often with little effect on the target pathogen . For example, antimicrobial administration to patients developing ventilator-associated pneumonia leads to loss of potentially protective species and proliferation of opportunistic pathogens , this is also true of antibiotic dependent C. difficile proliferation . Given that bacterial species are adept at sensing and responding to their environment, including microbial diversity, we are starting to appreciate, using culture-independent tools, how specific treatments impact the polymicrobial community and these shifts in community composition affect primary pathogen behavior. Comprehensively examining the effect of novel therapeutics on the microbial community diversity and function will yield unprecedented information on the effects of these treatments and provide valuable insight into unforeseen side-effects such as pathogen proliferation or unanticipated induction of specific virulence systems.
Development of therapeutics that target specific aspects of bacterial physiology, represent a promising approach to combating pathogenesis. Ultimately, understanding bacterial physiology at both the single species level and within the context of a dynamic mixed-species population in the human host will provide further therapeutic targets and potentially the means to manipulate and analyze whole communities behavior with the aim of reducing virulence gene expression, combating infection and improving host health.
SVL is funded by an American Lung Association award and a NIH award (AI075410). JWK is funded by NIH grants SCCOR HL 74005 and HL 69809, HL074005 (SCCOR Project 4).
*, special interest;
**, outstanding interest.