MINIREVIEW
Exploring the applications of invertebrate host–pathogen models for in vivo biofilm infections Sarah Edwards1 & Birthe V. Kjellerup1
IMMUNOLOGY & MEDICAL MICROBIOLOGY
Department of Biological Sciences, Goucher College, Baltimore, MD, USA
Correspondence: Birthe V. Kjellerup, Department of Biological Sciences, Goucher College, 1021 Dulaney Valley Road, Baltimore, MD 21204, USA. Tel.: +14103373019; e-mail:
[email protected] Received 1 December 2011; revised 13 April 2012; accepted 15 April 2012. Final version published online 21 May 2012. DOI: 10.1111/j.1574-695X.2012.00975.x Editor: Thomas Bjarnsholt Keywords invertebrate models; biofilms; poly-microbial infections; mono-microbial infections.
Abstract In the natural environment, microorganisms exist together in self-produced polymeric matrix biofilms. Often, several species, which can belong to both bacterial and fungal kingdoms, coexist and interact in ways which are not completely understood. Biofilm infections have become prevalent largely in medical settings because of the increasing use of indwelling medical devices such as catheters or prosthetics. These infections are resistant to common antimicrobial therapies because of the inherent nature of their structure. In terms of infectious biofilms, it is important to understand the microbe–microbe interactions and how the host immune system reacts in order to discover therapeutic targets. Currently, single infection immune response studies are thriving with the use of invertebrate models. This review highlights the advances in single microbial–host immune response as well as the promising aspects of polymicrobial biofilm study in five invertebrate models: Lemna minor (duckweed), Arabidopsis thaliana (thale cress), Dictyostelium discoideum (slime mold), Drosophila melanogaster (common fruit fly), and Caenorhabditis elegans (roundworm).
Introduction Development of simple model systems for the host–pathogen interaction has long been the investigative venture of many research projects. Owing to the similarities between human and invertebrate virulence modulators as well as infection pathogenesis, invertebrate models have become popular as less expensive, less ethically challenging, more efficient, and more genetically tractable hosts for the study of infection than the common murine models (Wilson-Sanders, 2011). Furthermore, unlike vertebrate models, invertebrate models negate the necessity of an Animal Care and Use Committee. Accordingly, in recent decades, there has been a thriving examination of the common threads of the mammalian and invertebrate immunity. Because the adaptive immune response is absent in invertebrates, the focus on innate immune function has led to profound developments for many invertebrate models. It is accepted that invertebrates show striking homology to mammalian innate immunity pathways, recognized and activated by Toll signaling, among others, in both humans and insects (Medzhitov et al., 1997). The response to the presence of pathogens is FEMS Immunol Med Microbiol 65 (2012) 205–214
an ancient response by invertebrates and vertebrates alike and so too are the methods by which such pathogens launch their attack. It is now recognized that pathogens contain several highly conserved genes that regulate the expression of virulence factors in both plants and animals (Rahme et al., 1995). For these reasons, the use of invertebrate models has become attractive in investigating the relationship between pathogen and the host innate response. However, there are facets about such models that fail to simulate true infection environments, particularly the polymicrobial biofilm infection. A biofilm is a microbial community encased in a self-produced polymeric matrix which, if it is polymicrobial, can contain both fungi and variable bacterial consortia (Costerton et al., 1995). According to early studies of biofilms, they can cause chronic systemic infections by sloughing off parts of this community while the rest can remain resistant to antimicrobial therapies (Costerton et al., 1995; Chandra et al., 2001). Biofilms have been found colonizing indwelling medical devices that are ubiquitous in modern medicine such as central venous catheters, urinary catheters, pacemakers, and mechanical heart valves (Donlan, 2001). ª 2012 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved
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Fungal biofilms of Candida albicans are found to be significant contributors to nosocomial bloodstream and systemic infections (Kuhn & Ghannoum, 2004). It has been demonstrated that 27% of Candida bloodstream infections are polymicrobial (Klotz et al., 2007); however, it has not been verified whether the point source is a polymicrobial biofilm. Still, Harriot & Noverr (2009) found that the polymicrobial biofilm formed by C. albicans and Staphylococcus aureus demonstrated enhanced resistance by S. aureus to a general antibiotic, vancomycin. Also, in a study by Carlson & Johnson (1985) in mice, it was shown that colonies of S. aureus always were found in association with C. albicans and within the fungal growth, when these pathogens were observed by microscopy in the abdominal cavity of mice (Carlson & Johnson, 1985). A few years earlier, the same author discovered that this co-inhabitation caused 100% mortality compared to very low or no mortality, when the pathogens were present individually in the murine abdomen (Carlson, 1982). These results would likely today be recognized as biofilms in the abdominal cavity. In spite of frequent co-isolation of several pathogenic species from an infection site, identification of polymicrobial clinical isolates does not guarantee the presence of a polymicrobial biofilm, since visual inspection is needed to identify that the involved species are truly encased in the same matrix (which is the definition of a biofilm). This has for instance been observed in sputum samples from chronically infected patients with cystic fibrosis (CF), where monomicrobial biofilms consisting of Pseudomonas aeruginosa were detected by microscopy in explanted lungs (Bjarnsholt et al., 2009; Rudkjobing et al., 2011). In the study by Rudkjobing et al., it was also found that non-endstage patients with CF harbored heterogeneous microbial communities. However, the identified bacteria were all found to exist in monospecies aggregates (Rudkjobing et al., 2011). A similar finding was reported in chronic wounds from patients, where S. aureus and P. aeruginosa were identified in many of the wounds by culturing. As it was the case with the CF patients, micro-scopy revealed the presence of P. aeruginosa monomicrobial biofilm aggregates embedded in matrix material (alginate), whereas S. aureus could not be observed simultaneously in this matrix (Kirketerp-Moller et al., 2008). Thus, these findings show that polymicrobial biofilm infections have not yet been unambiguously observed in patients, where so far only monospecies biofilm infections have been documented. However, polymicrobial biofilm infections have been developed in several mammalian host models, and considering the current rise of bacterial resistance because of the overuse and/or misuse of antimicrobial therapies, it would be beneficial to understand as much as possible about polymicrobial interactions within a biofilm. ª 2012 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved
S. Edwards & B.V. Kjellerup
Despite their prehistoric coexistence, little is known today about inter-species/kingdom interactions within a biofilm environment or the mechanisms behind their resistance to common therapeutic techniques. In vitro studies have shown that bacterial–fungal interactions enhance the pathogenicity of the biofilm through bacterial–hyphal attachment (Hogan & Kolter, 2002). A study conducted with S. aureus and C. albicans not only documented the enhanced adherence of the bacteria to fungal hyphae, decreasing its necessity to compete for host surface adherence, but also showed the use of differential protein expression, indicating that C. albicans may increase S. aureus virulence (Peters et al., 2010). Yet, another study showed that Acinetobacter baumannii targets the complexes necessary for C. albicans biofilm formation (Peleg et al., 2008). Although we know of the existence of polymicrobial biofilms, little is documented about the microbial relationships, whether they are synergistic, commensal, or antagonistic. Additionally, although providing vital information as to the interaction between the pathogens, these few studies fail to elucidate the connection between these activities and the host innate immune response. Here, we propose that a facile in vivo model should be developed to simulate the complexity of the polymicro-bial biofilm while allowing the immune response to be simplified by comparison of human immunity to what is available in the model host. All chosen models lack adaptive immune systems, which are activated during chronic infections in humans (Jensen et al., 2010), and thus, a developed model would simulate early-stage infections in vertebrates. Because of their contributions to host–microbe interaction studies, the hosts Lemna minor (duckweed), Arabidopsis thaliana (thale cress), Dictyostelium discoideum (slime mold), Drosophila melanogaster (common fruit fly), and Caenorhabditis elegans (roundworm) will be discussed as the candidates for model hosts. These models will be described and evaluated with regard to their relevance for polymicrobial biofilm infections.
Comparative models Lemna minor
In terms of host–pathogen models, L. minor (duckweed) is one of the simplest model organisms. This water-dwelling plant, which normally reproduces by budding, rapidly generates genetically consistent clones, making it a reliable scientific model (Brain & Solomon, 2007). The ease of its handling, inexpensiveness of its maintenance, and its sensitivity toward both organic and inorganic chemicals are also attractive features of duckweed FEMS Immunol Med Microbiol 65 (2012) 205–214
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(Table 1). As reported by Zhang et al., duckweed was inoculated with various pathogens including P. aeruginosa, Escherichia coli, and S. aureus as well as several clinical isolates of other highly pathogenic bacteria. In these experiments, one duckweed plant was placed into a cell culture well and inoculated with an overnight culture of the respective bacterium and grown for 5 days. Afterward, the plants were analyzed based on fresh weight and chlorophyll content, and it was noticed that biofilm formation occurred on both the leaves and the roots (Zhang et al., 2010). This virulence assay concluded that fresh weight and chlorophyll content was reduced when affected by bacterial strains with increased pathogenicity (Zhang et al., 2010). Furthermore, the effect of antimicrobials was demonstrated successfully using this model by evaluating the decreased effects of the pathogens on the plant. The use of mutant strains indicated that P. aeruginosa strains, either mutant in quorum-sensing (QS) systems or containing human serum paraoxonase known to inactivate P. aeruginosa QS signals, were less virulent than the wild type (Primo-Parmo et al., 1996; Zhang et al., 2010). Additionally, duckweed reacted negatively toward a pathogenic strain of S. aureus, whereas it was unaffected by the nonpathogenic strain (Zhang et al., 2010). Application of this simple model is an exciting step for the evaluation of the host–pathogen relationship because it simplifies and eliminates many variables that are difficult to interpret with more complex models, thus allowing the research to focus on specific questions. The potential of duckweed is great in terms of elucidating the mechanisms behind pathogenicity through the use of mutants and molecular analyses; however, this model is still in its infancy, and its genetics in regard to immunity has yet to be clearly connected to vertebrate hosts. Arabidopsis thaliana
Arabidopsis thaliana (thale cress) has long been used as an in vivo host system for plant and animal physiological and biochemical studies (Ward, 2001; Hays, 2002). Within recent years, it has become an attractive model for virulence studies (Table 1). Several human pathogens considered to be highly pathogenic, especially in hospital environments where immunocompromised patients are abundant, have been found to also infect A. thaliana (Prithiviraj et al., 2005a, b). The A. thaliana root model has proved useful in the investigation of infection and/or biofilm formation of pathogens including P. aeruginosa, Enterococcus faecalis, and S. aureus, three of the most threatening bacteria in hospital settings (Prithiviraj et al., 2005a). Through virulence assays in which Arabidopsis leaves were injected with bacteria, it was found that the human isolate of P. aeruginosa PA14 caused severe plant FEMS Immunol Med Microbiol 65 (2012) 205–214
rot in some ecotypes of A. thaliana (Rahme et al., 1997). Similar results were also obtained by infection with the Arabidopsis pathogen Pseudomonas syringae (Rahme et al., 1995). Several virulence factors were demonstrated to be active in both plant and animal models including toxA, plcS, and gacA (Rahme et al., 1995). These results were validated through screening of random TnphoA mutant PA14 isolates, nine of which were found to have attenuated virulence in both A. thaliana plant rot and mouse burn models (Rahme et al., 1997). The pathogenesis of PA14 on A. thaliana, which has since been described in detail, involves the formation of biofilm-like structures in the colonization process (Plotnikova et al., 2000). Little has been found with regard to enterococcal virulence factors with the use of cumbersome mammalian models (Jha et al., 2005). However, by following mutant screens of virulence factors analogous to those utilized by E. faecalis in mammalian systems such as the QS system (DfsrB) in simple hosts, Jha et al., 2005; found a positive correlation between the number of cells on infected A. thaliana roots, biofilm formation, and the virulence of the infection. With regard to S. aureus biofilm in the A. thaliana root infection model, a host abundance of salicylic acid was found to attenuate the virulence (Prithiviraj et al., 2005a, b). It is yet unknown whether this attenuation of several strains of S. aureus was a result of the direct effect of salicylic acid or whether it is a host-response to the abundance of salicylic acid (Prithiviraj et al., 2005a, b). These studies exemplify how the use of simple models could be more advantageous than higher animals mostly due to the fact that high-throughput mutant screening is a more convenient technique with invertebrate models than with vertebrates (Rahme et al., 1991). Furthermore, a complete mitogen-activated protein kinase (MAPK) cascade has been determined as a highly conserved mechanism involved in the pathogen-associated molecular pattern response of the plant A. thaliana, giving this model even more relevance toward discovering novel aspects of mammalian innate immunity (Asai et al., 2002). Such studies have demonstrated the significant overlap in pathogenesis of several human pathogens to plants, including biofilm infections, but there are still no studies attempting to examine the polymicrobial effects on pathogenesis. In this way, the A. thaliana root-biofilm model shows great potential, but should be extended to include multiple microbial pathogens, spanning both species and kingdoms. Dictyostelium discoideum
The model organism, D. discoideum (slime mold), a common haploid soil ameba, may seem like an unlikely ª 2012 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved
ª 2012 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved
30 °C
Leaf dip or injection, root coculture Yes
5 days
28 °C
Coculture
5 days
Zhang et al. (2010)
Unknown
No
Rahme et al. (1995, 1997), Prithiviraj et al. (2005a, b)
MAP kinase cascade
Light
Solomon & Isberg (2000), Steinert & Heuner (2005), Bozzaro & Eichinger (2011)
Human macrophages
3–10 days*
Yes
Feeding assay, coculture
24.5 °C
Social, spore generation
24 h
HLS media
Bacteria
Slime mold
Dictyostelium discoideum
De Gregorio et al. (2002), Tzou et al. (2002), Lemaitre & Hoffmann (2007), Lutter et al. (2008), Sibley et al. (2008), Apidianakis & Rahme (2009), Mulcahy et al. (2011)
Toll, Imd, AMP, JAL/STAT
12–36 h
Yes
Needle pricking, injection, feeding assay
25 °C
Sexual
Roughly 10 days
Standard cornmeal sucrose medium, 25 °C, 65% humidity, 12-h light cycle
Standard cornmeal sucrose medium
Common fruit fly
Drosophila melanogaster
Kim et al. (2002), Mylonakis et al. (2003), Mylonakis & Aballay (2005), Sifri et al. (2006), Begun et al. (2007), Tenor & Aballay (2008)
Toll-like receptors, p38 MAPK
2 days–1 month (depending on the strain of C. elegans)
Yes
Feeding assays
16–25 °C
Hermaphroditic
4–7 days
Nematode growth medium, E. coli OP50, 16–25 °C
Bacteria
Roundworm
Caenorhabditis elegans
Summary of the information provided as reference for the models discussed in the text. All models lack adaptive immunity, and thus, the infection being studied is more relevant to early-stage infection involving innate immune response. *Studies conducted on Dictyostelium discoideum were based on the consummation of a bacterial lawn (biofilm).
Known relevance to mammalian immunity References
Mode of reproduction Model infection temperature range Infection assay technique Complete genome sequence Length of biofilm infection assay
Budding, cloning
Growth media/ conditions
Generation time
Peat mix for leaf and soil studies, MS basal media for root studies 3 weeks in soil, 2 weeks in media Sexual
Light
Schenk & Hildebrandt (SH) basal medium supplemented with 1% sucrose 1 week
Food source
Thale cress
Duckweed
Common name
Arabidopsis thaliana
Lemna minor
Model characteristics
Table 1. Reviewed invertebrate models
208 S. Edwards & B.V. Kjellerup
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candidate for the host–pathogen model. Nevertheless, recent research has found it to be useful for modeling several human bacterial and fungal pathogens (Table 1) (Bozzaro & Eichinger, 2011). Thus far, P. aeruginosa, Cryptococcus neoformans, Mycobacterium spp., and Legionella pneumophila have been studied in D. discoideum with success (Steinert & Heuner, 2005). In practice, the ameba is harvested from a growth medium and incubated with the respective bacteria or fungi at 27 °C, a critical temperature above which the D. discoideum cannot survive, and then monitored for bacterial–fungal reproduction within the host cell over time (Solomon & Isberg, 2000). This ‘invasion assay’ was performed with aforementioned bacterial species, where the slime molds parallel the human macrophage invasion by pathogens and have provided critical evidence in terms of conserved pathogenesis. For example, the coculture of Mycobacterium avium and D. discoideum causes cell lysis of the ameba from a 100-fold intracellular growth increase by M. avium, a phenomenon also observed with human macrophages (Skriwan et al., 2002). Mycobacterium avium is a common contaminant in hospital waters of developing countries wreaking havoc to immunocompromised patients. It has also been demonstrated that P. aeruginosa utilizes conserved virulence factors to infect D. discoideum (Pukatzki et al., 2002). A simple plating assay was developed in which ameba is introduced to bacterial lawns (biofilm) and incubated over a number of days. If plaques are observed from amebae consumption of bacterial lawn at this time, the bacteria are considered nonpathogenic. No plaques were observed when D. discoideum was incubated with P. aeruginosa strain PA14, indicating that the strain was highly virulent toward the ameba. It was also deduced that PA14 kills D. discoideum through two pathways: the QS mechanism as well as a type-III secretion system (Pukatzki et al., 2002). This simple plaque assay was most recently employed to discover that the swine pathogen, Streptococcus suis, is also pathogenic toward D. discoideum, although the virulence factors are still uncertain (Bonifait et al., 2011). Since the publishing of the complete genome of D. discoideum in 2005, there has been little advancement toward discovery of novel virulence factors or host innate response mechanisms, leaving ample opportunity for model development (Eichinger et al., 2005). Moreover, there are limitations to this model that must not be overlooked, the most important being that D. discoideum cannot survive above 27 °C, and therefore, many of the virulence factors normally expressed in humans at 37 °C cannot be studied. This is a common setback for many invertebrate models, including the ones mentioned in this study; however, D. discoideum is maintained at the lowest FEMS Immunol Med Microbiol 65 (2012) 205–214
temperatures of all mentioned models. With these factors in mind, this may not be a well-suited candidate for a polymicrobial biofilm host system at present, but it might have future potential. Drosophila melanogaster
One of the most well-studied biological host systems is the fruit fly species, D. melanogaster. Its genome is thoroughly known, enabling the identification of microbial response factors, and its ease of manipulation allows for broad mutant screening (Apidianakis & Rahme, 2009). Aside from its ubiquitous use in molecular biology, research with D. melanogaster has exhibited exceptional results in the realms of microbiology and immunology as well, with both the discovery of virulence factors and innate host immune response (Lemaitre & Hoffmann, 2007; Apidianakis & Rahme, 2009). Most recently, several important infection models including Burkholderia cepacia, P. aeruginosa, S. aureus, E. faecalis, C. albicans, and C. neoformans have been developed in D. melanogaster (D’Argenio et al., 2001; Alarco et al., 2004; Apidianakis et al., 2004; Needham et al., 2004; Cox & Gilmore, 2007; Apidianakis & Rahme, 2009; Castonguay-Vanier et al., 2010). Currently, three basic modes of infection with this model exist: (1) the pricking, which resembles a wound infection; (2) the injection of a finite amount of bacterial or fungal cells, which simulates a systemic infection; (3) the feeding assay, which mimics an intestinal infection (Apidianakis & Rahme, 2009). One study reported differences between the results obtained from feeding and injection techniques using P. aeruginosa isolated from burn wounds and CF (Lutter et al., 2008). Moreover, a recent study has proved D. melanogaster as being an appropriate host for P. aeruginosa biofilm infection (Mulcahy et al., 2011). Aside from single pathogen models, the D. melanogaster model has made the crossover into polymicrobial infection studies (Sibley et al., 2008). For example, patients with CF have lungs that are often chronically infected by opportunistic pathogens whose interactions with other normal bacterial inhabitants lead to more virulent infections (Duan et al., 2003). Through modified feeding assays, recent research has examined the interaction between 40 oropharyngeal isolates of patients with chronic CF and P. aeruginosa strain PA01 (Sibley et al., 2008). It was found that the 40 isolates could be divided into three classes: (virulent) able to kill on their own but faster with PA01, (synergistic) unable to kill on their own but kill in the presence of PA01, and (avirulent) unable to kill with or without PA01 (Sibley et al., 2008). Additionally, by a CTX (Cholera toxin lysogenic bacteriophage) integration system, several virulence factors ª 2012 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved
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including those involved in QS and motility were found to be upregulated, while several host innate immunity targets were implicated to have been involved in the cause of death (Sibley et al., 2008). The findings from this model support previous in vitro studies showing that gene regulation of virulence factor expression can be altered within a polymicrobial infection and demonstrates the necessity for in vivo studies to understand the hostresponse (Peters et al., 2010). While the host-response of D. melanogaster to polymicrobial infections may not be completely understood, several major pathways in their innate immunity have long been accepted to regulate the expression of antimicrobial peptides (AMPs), the most relevant being the JAK/STAT, Imd, and Toll pathways (Table 1) (Lemaitre & Hoffmann, 2007). Although little is known about the initiation and downstream effects of the JAK/STAT pathway, the Imd and Toll pathways have been demonstrated to have striking similarities to the vertebrate activation of the NF-jB factors, which are essential components in the human immune response (Tzou et al., 2002). In Drosophila, Toll is activated by gram-positive bacteria, fungi, and yeast, whereas Imd is activated by gram-negative bacteria (De Gregorio et al., 2002). Drosophila innate immunity has been reviewed in detail, making it an accepted model for polymicrobial interaction (Lemaitre & Hoffmann, 2007). Caenorhabditis elegans
Since Sydney Brenner’s inaugural study of C. elegans as a useful model system for genomic and developmental investigations, they have become a common vessel for research of all kinds (Mylonakis et al., 2003). Their short generation time, large number of offspring, complete genetic tractability, transparent cuticle, and wide array of developed mutants have made these soil-derived nematodes a popular model host system for various biological niches including apoptosis and organ development, RNA interference, and GFP expression, all of which have led to Nobel Prize–winning research (Mylonakis et al., 2003). In the most recent decades, the application of C. elegans has expanded dramatically in microbiology, first being used to model the host–pathogen interaction with P. aeruginosa, which was found to be pathogenic to C. elegans (Tan et al., 1999b). The methodology of the model has been heavily explored. At its most basic, C. elegans are reared on nematode growth media seeded with E. coli, OP50. Unless the particular strain of C. elegans calls for something else, the nematodes can be grown at room temperature, making their maintenance easy. Typically, the adult stage (L4) is washed and picked onto a plate of ª 2012 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved
S. Edwards & B.V. Kjellerup
the respective pathogenic bacteria or fungi to be tested. The nematodes are counted for survival over time and considered dead when unresponsive to touch (Kurz & Ewbank, 2000). Through this technique, a multitude of pathogens have been studied and categorized according to their killing tactic as ‘slow killing’ or ‘fast killing’ (Mylonakis et al., 2003). Fast killing would indicate a toxin-mediated attack, whereas slow killing would signify an infection. To distinguish the fast-killing method, C. elegans killed by bacterial cultures that have undergone heat-killing or UV-radiation suggest that fast killing can be accomplished by heat-stable toxins (Tan et al., 1999a, b). The slow killing is demonstrated by counting colony-forming units (CFUs) recovered from a dead host, which are indicative of intestinal infection (Mylonakis & Aballay, 2005). Additionally, when feeding C. elegans a mixture of bacteria, killed nematodes can be ground and plated to examine and compare relative amounts of CFUs formed by the different bacterial species in the feed (Garsin et al., 2001). This technique supports our suggestion for the use of C. elegans to study polymicrobial infections. Once the model proved useful in the discovery of virulence factors initially using a P. aeruginosa PA14:TnphoA mutant screen to ascertain several important virulence factors’ – toxA, gaxA, and lasR – involvement in P. aeruginosa pathogenicity toward C. elegans, it was also tested on a multitude of other pathogens (Tan et al., 1999a, b). The results showed that many of the most pertinent human pathogens have been found to also kill C. elegans such as several strains of Enterococcus, Staphylococcus, Salmonella, Burkholderia, Serratia marscescens, as well as C. albicans and C. neoformans (Aballay et al., 2000; Kurz & Ewbank, 2000; Tan & Ausubel, 2000; Garsin et al., 2001; O’Quinn et al., 2001; Gan et al., 2002; Mylonakis et al., 2002; Sifri et al., 2003; Peleg et al., 2008). Extensive listings of human pathogens with the potential to kill C. elegans have been compiled with proposed virulence factors and mediators of pathogenicity (Sifri et al., 2006). Additionally, owing to the transparent nature of the nematode cuticle, green fluorescent protein or fluorescently tagged organisms can be easily visualized through both fluorescent and contrast microscopy without manipulation of the nematode itself (Tan et al., 1999a, b). In this way, C. elegans is a competitive model for the study of host–pathogen interaction in terms of single pathogen infection. Recent C. elegans research has crossed over into the study of biofilm and polymicrobial systems. One study has found that Staphylococcus epidermidis, an opportunistic and multiresistant pathogen known for its prevalence in nosocomial infections, demonstrates enhanced virulence when it is present in a biofilm within the gut of the nematode (Begun et al., 2007). This supports C. elegans FEMS Immunol Med Microbiol 65 (2012) 205–214
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Invertebrate models for biofilm infections
as a novel model for the study of host–biofilm interactions. Also in support of using C. elegans as a model for microbe–microbe interactions, it has been found through screening of E. faecalis strains co-infecting with pathogenic E. coli that a synergistic virulence took place when both bacteria were inoculated compared to either one alone (Lavigne et al., 2008). Results such as these give insight into the role that Enterococci might play within a polymicrobial environment and provide a basis for the methodology by which other pathogens may be tested. Furthermore, the relationship between prokaryotic and eukaryotic pathogens has also been investigated using C. elegans as a model organism for infection. In observing the interaction between the ubiquitous opportunistic fungus, C. albicans, and the multiresistant bacterium A. baumannii, it was noticed that until a quorum was developed by C. albicans, the interaction was antagonistic, indicating that A. baumannii possessed antifungal properties (Peleg et al., 2008). Once a biofilm was developed in which QS molecules can be activated, those properties ceased to effect the growth of C. albicans. Here, Peleg et al. even suggest that polymicrobial studies which include the investigation of microbial synergy and pathogenesis may lead to possible therapeutic targets on, for example, QS molecules. These studies extend the model into the polymicrobial setting and make the study of dual infections an attainable goal. As with many other models, the largest challenge is human relevance. Just as C. elegans has been successful in providing novel information in terms of pathogen virulence factors, they have also been useful in the discovery of host-response mechanisms (Mylonakis & Aballay, 2005). While C. elegans, like all invertebrate models, lack an adaptive immune system, their innate immunity has been ambitiously investigated. Several pathways involved in innate immune activation and AMP synthesis are of high interest in C. elegans host–pathogen research (Tenor & Aballay, 2008). The most important pathway involved in C. elegans innate immunity, deduced through the employment of mutant nematodes, is the p38 MAP kinase signaling cascade, for which activity is conserved in mammalian systems (Table 1) (Kim et al., 2002). Unlike in Drosophila which depends on Toll recognition of microbial pathogens, C. elegans possesses Toll homologs, but they are not active in their innate immune process (Pujol et al., 2001; Mylonakis & Aballay, 2005). Recently, Toll-like receptors (TLRs) have been indicated in activating defensin-like proteins in response to Salmonella enterica, providing evidence that TLRs may be evolutionarily conserved (Tenor & Aballay, 2008). Such gains in the relatively modest area of C. elegans innate immunity support the model’s validity as a vessel for the study of host–pathogen interactions. FEMS Immunol Med Microbiol 65 (2012) 205–214
Conclusions The knowledge of microbial pathogenesis has been greatly augmented by the successes of multiple invertebrate models. They have facilitated the discovery of novel virulence factors and provided insight into the innate immune system where, in some cases, the use of murine models would have proved more difficult. However, few models have extended their technique to include polymicrobial infections. Just as the methods for single pathogen study require the use of mutant screening, polymicrobial studies will employ even more mutant screens, thus demanding a genetically tractable fast reproducing model to yield accurate results. Invertebrate models can serve this function and act as a stepping-stone on which to isolate certain virulence or host immune factors to focus on using vertebrate models. Temperature limitations are apparent in each of these models; thus, any positive validation from a vertebrate system would indicate the independence of a virulence factor from temperature. As of now, duckweed is novel and provides rapid results for virulence and antimicrobial studies. A complete genome sequence of duckweed would enhance the model greatly, allowing for the study of immune factors. Arabidopsis is an easy model to conduct mutant screening pathogenicity assays for both leaf injections and root-biofilm models. However, polymicrobial studies have yet to be performed on this model. The social ameba, D. discoideum, has been infected by several human pathogens, but its immune response has only been superficially explored. Drosophila melanogaster and C. elegans are both well developed with powerful genetics, easy and inexpensive maintenance, and a plethora of common pathogens to humans. Both models have reached into polymicrobial studies, and thus, the techniques are relatable for the study of polymicrobial biofilms. Furthermore, each model possesses several known innate pathways that have been related to vertebrate models. In terms of the workability of each model, the techniques devised for C. elegans are more efficient than for Drosophila. Where much time is spent on the mode of infection in Drosophila for either pricking or injection, C. elegans are left to feed on a lawn of bacteria or fungi. Additionally, C. elegans have a transparent cuticle allowing for more powerful microscopy and fluorescent techniques. Moreover, techniques for modeling single microbial biofilms in C. elegans have been developed; thus, methods have already been established using strains that represent various degrees of immune function. For models in which the immune response has not yet been fully exposed, like duckweed, initial colonization studies could be inexpensively conducted to understand necessary microbe–microbe interactions for biofilm development. As we suspect that these interactions within a polymicrobial biofilm setting might play an ª 2012 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved
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important role in increased virulence, tolerance, and resistance of infectious biofilms, the host model should be useful in the identification of a common matrix to verify the manner of contact the microorganisms have within the infection site. Although it is possible to extend each of these models to study polymicrobial biofilms, C. elegans would be the most thorough model to elucidate both virulence factors and host innate immune functions and to visualize the infection.
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