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Crop integrity in response to P. aeruginosa infection. (A) The macroscopic structure of (A), uninfected and (B), PAO1pCHAP6656- infected Drosophila crops (Olympus OV100 intravital observation system). Merged fluorescent image of phallodin 488-stained actin (green) and DAPI- stained nuclei (blue) in uninfected crops using (C) 10x and (D) 63x objectives. PAO1pCHAP6656-infected crops (red) at (E) low and high (F) magnification. Scale bars in C and E indicate 400 m M; scale bars in D and E indicate 100 m M. White arrows in C and E indicate the area of the crop where higher magnification images were taken. At least five infected crops were examined from two separate infections and representative images are shown. doi:10.1371/journal.ppat.1002299.g004 

Crop integrity in response to P. aeruginosa infection. (A) The macroscopic structure of (A), uninfected and (B), PAO1pCHAP6656- infected Drosophila crops (Olympus OV100 intravital observation system). Merged fluorescent image of phallodin 488-stained actin (green) and DAPI- stained nuclei (blue) in uninfected crops using (C) 10x and (D) 63x objectives. PAO1pCHAP6656-infected crops (red) at (E) low and high (F) magnification. Scale bars in C and E indicate 400 m M; scale bars in D and E indicate 100 m M. White arrows in C and E indicate the area of the crop where higher magnification images were taken. At least five infected crops were examined from two separate infections and representative images are shown. doi:10.1371/journal.ppat.1002299.g004 

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Pseudomonas aeruginosa is an opportunistic pathogen capable of causing both acute and chronic infections in susceptible hosts. Chronic P. aeruginosa infections are thought to be caused by bacterial biofilms. Biofilms are highly structured, multicellular, microbial communities encased in an extracellular matrix that enable long-term survival in the...

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... Prior to this study, it was not known if the fly immune system responded differently to biofilm and non-biofilm forming bacteria. Drosomycin expression is regulated through the Toll pathway [64]; Diptericin is regulated via the Imd pathway [65] and both pathways overlap to regulate cecropin A1 expression [66]. Our data indicates that both of the central immune pathways in Drosophila are activated in response to biofilms. In addition this data indicates that it is Pel positive biofilms, and possibly Pel EPS itself, that may act as a specific host immune signal inducing AMP gene expression in Drosophila as a psl mutant has no effect on Drosophila killing (Figure S3). Future work will focus on identifying the specific bacterial components involved in AMP gene expression and other host signalling pathways in response to Pel and Psl positive biofilms and non-biofilm P. aeruginosa infections. In the Drosophila oral infection model, our data suggests that Pel positive biofilms induced AMP gene expression in the fly. Although biofilm infections induce AMP gene expression (Figure 8A–C), biofilm-forming bacteria isolated from fly crops postinfection are more resistant to the AMPs polymyxin B and colistin than those recovered from planktonic cultures (Figure 6). Bacterial Pel EPS may be a cue to the host to increase AMP gene expression thus serving to slow dissemination of the bacteria, and in this way slow systemic infection which would rapidly kill the host. On the other hand, EPS may also induce inflammation in the crop/GI system resulting in a localized damage to the host. Strains incapable of forming Pel positive biofilms in vivo resulted in a decreased AMP response but disseminated earlier, resulting in a systemic infection associated with faster host killing. These interpretations are supported by the Drosophila survival data obtained from co-infection experiments, where co-infection of flies with pelB::lux and PAO1 significantly increases Drosophila survival compared to infection with pelB::lux alone. It has previously been shown that P. aeruginosa eludes host defenses by suppressing AMP gene expression in a Drosophila model of acute infection [24]. This study also demonstrated that infection with a less virulent P. aeruginosa strain resulted in immune potentiation and protected flies from subsequent acute infection with a more virulent P. aeruginosa strain [24]. To determine if oral infection, biofilm formation and induction of AMPs in Drosophila could alter the kinetics of fly survival following subsequent acute infection, we performed the following experiment. Male flies were orally infected with PAO1 (biofilm, AMP induction), pelB::lux (non- biofilm, AMP repression) or PAZHI3 (hyperbiofilm, AMP induction) for 24 h. After 24 h, orally infected flies from each of the three groups above and uninfected flies were nicked with PAO1 (acute infection), LB (sterile nicking) or not treated. Oral infection with PAO1, pelB::lux or PAZHI3 had no significant effect on the rate of fly survival during subsequent acute infection (nicking) with PAO1 (Figure 9). To determine if oral PAO1 or PAZH13 biofilm infections altered Drosophila survival following subsequent oral infection with pelB::lux, the following experiment was performed. Drosophila were allowed to feed on PAO1, PAZH13, pelB::lux or a sucrose control for 24 h (primary infection), which is sufficient for biofilm formation to occur in the crop (Figure 1). After 24 h, all flies were transferred to new vials containing pelB::lux as the food source (secondary infection). Survival was monitored up to 14 days after the primary infection. Primary infection with PAO1 or PAZH13, followed by secondary infection with pelB::lux significantly increased fly survival compared to flies who were infected with pelB::lux for both the primary and secondary infection. Increased Drosophila survival following primary infection with PAO1 or PAZH13 was not due to failure of the secondary infecting pelB::lux strain to infect Drosophila, as pelB::lux tetracycline resistant colonies (the antibiotic marker of the lux transposon) were recovered (at $ 3.8 6 10 6 CFU/fly or 76–99% of total bacterial load) from all secondary pelB::lux infected flies 5 days postinfection. Primary oral infection with a biofilm-forming strain protected Drosophila from secondary oral infection with pelB::lux . Oral infection with a biofilm forming strain induced AMP gene expression, which may explain why increased fly survival was observed against secondary oral infection with pelB::lux . However the AMPs induced following oral infection may not be sufficient to alter Drosophila survival against subsequent acute infection. A possible reason for this is that AMP induction following biofilm infection is localized to the gut and does not protect Drosophila from death as a result of pricking and acute systemic infection. It is also possible that the pathology resulting from tissue damage following oral infection (Figure 4) may prevent Drosophila from responding to and coping with subsequent acute infection. P. aeruginosa infections are associated with the highest case fatality rate of all Gram-negative infections [67]. This is partly due to the ability of P. aeruginosa to resist antimicrobial therapy. One of the main evasion strategies used by P. aeruginosa , and other microbes, is the formation of multicellular, dense aggregates called biofilms. We have shown that specific antibiotic resistance mechanisms are induced in P. aeruginosa biofilms [4]. Biofilm infections are estimated to account for 65% of all bacterial infections [68]. While some studies have investigated the host response to P. aeruginosa infection [20,69,70], little is known regarding the bacterial and/or host factors involved in the pathogenesis of biofilm infections. The aim of this research was to develop a Drosophila infection model that enables biofilms to be intricately studied in vivo . In this work we present evidence that oral infection of Drosophila by P. aeruginosa PAO1 resulted in biofilm formation in the Drosophila crop (Figure 1). We demonstrated that biofilms formed in vivo retain the typical characteristics of in vitro grown biofilms, including DNA and EPS staining (Figure 2) and increased resistance to antibiotics (Figure 6C). We also showed that biofilm infections resulted in significantly decreased numbers of bacteria disseminating to the hemolymph 2 days postinfection, and contributed to increased AMP gene expression in the fly (Figures 6, 8). Non-biofilm forming pelB::lux infections, on the other hand, resulted in decreased AMP gene expression in the fly, significantly increased numbers of bacteria disseminating to the hemolymph 2 days postinfection, as well as early and increased fly mortality (Figures 6–8). The increased virulence of the pelB::lux mutant was attenuated by co-infection of Drosophila with biofilm-forming and AMP-inducing strains PAO1 or PAZH13 (Figure 8D). Furthermore, primary infection with either of these AMP-inducing strains altered the survival kinetics of Drosophila from secondary oral infection with the more virulent pelB::lux but not from subsequent acute infection (Figure 9). In summary, we have developed a novel P. aeruginosa biofilm model of infection that can be used for studying both the bacterial and host response during infection. This model has the potential to significantly increase our understanding of the relationship between biofilms and the host during infection and also to tease out fundamental differences between the host response to biofilm and non-biofilm P. aeruginosa infections. Pseudomonas aeruginosa PAO1 and PAO1::p16Slux [63] were used as wildtype P. aeruginosa strains. The pelB::lux mutant is from a mini- Tn5- lux transposon mutant library that was previously constructed and mapped [48]. PAZHI3 is an rsmA mutant in the PAO1 background [61]. The plasmid pCHAP6656 encodes mCherry fluorescent outer membrane-anchored lipoproteins [42]. Drosophila were maintained routinely on medium containing corn meal, agar, sucrose, glucose, brewers’ yeast, propionic acid, and phosphoric acid [71]. Infections were performed as previously described [25]. Mid-log phase LB cultures of P. aeruginosa were spun down and resuspended in 5% sucrose. Cultures were adjusted to an OD600 = 25 (2.5 6 10 10 CFU per ml) in sucrose. The resuspended cells (0.12 mls) were spotted onto a sterile filter (Whatman) that was placed on the surface of 5 ml of solidified 5% sucrose agar in a plastic vial (VWR). The vials were allowed to dry at room temperature for approximately 30 minutes prior to addition of Drosophila . Because of the high concentration of bacteria on the feeding discs and the possibility of bacteria forming aggregates on the feeding discs over time, male Canton S flies (1–3 days old) were starved for 3 hours prior to being added to vials (10–14 flies per vial). This ensured that Drosophila fed heavily on P. aeruginosa within the first couple of hours. It is therefore unlikely that the P. aeruginosa strains on the filters had sufficient time to form biofilms prior to being eaten by Drosophila and causing an infection. Male flies were used as the infection lasts up to 14 days. During this time period females would have laid eggs, which if hatched, would interfere with the experimental results. Flies were anaesthetized by placing them on an ice-cold tile throughout the sorting and transferring process. Infection vials were stored at 26 u C in a humidity controlled environment. The number of live flies to start the experiment was documented and live flies were counted at 24 hour intervals. Healthy 3 day-old male flies were used in the fly nicking assays according to a modified method of [20]. Flies were sorted following anesthesis on a cold tile. The male flies were nicked in the dorsal thorax with a 27.5-gauge needle (BD Biosciences), which was dipped in bacterial culture normalized to an optical density at ...

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... Research on in situ and in vivo biofilms remains time-consuming and expensive. Some studies have used non-mammalian models as alternative hosts, such as Drosophila melanogaster [151,152] and Caenorhabditis elegans [153] to investigate biofilm infections and intestinal colonization in vivo. ...
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... To corroborate the results in the C. elegans survival assays, we next examined host survival in an established D. melanogaster oral infection model developed to study P. aeruginosa biofilm infections in vivo. The advantage of this model is that the innate immune system of Drosophila has similarities with the vertebrate innate immune system, thus aiding our understanding the role PelA's hydrolase activity may play in virulence in mammals 48 . As a negative control, Drosophila were fed 5% (w/v) sucrose. ...
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