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Pathogenesis of plant-associated Pseudomonas aeruginosa in Caenorhabditis elegans model

Background Pseudomonas aeruginosa is a globally dreaded pathogen that triggers fatality in immuno-compromised individuals. The agricultural ecosystem is a massive reservoir of this bacterium, and several studies have recommended P. aeruginosa to promote plant growth. However, there were limited attempts to evaluate the health risks associated with plant-associated P. aeruginosa. The current study hypothesized that agricultural P. aeruginosa strains exhibit eukaryotic pathogenicity despite their plant-beneficial traits. Results We have demonstrated that feeding with the plant-associated P. aeruginosa strains significantly affects Caenorhabditis elegans health. Out of the 18 P. aeruginosa strain tested, PPA03, PPA08, PPA10, PPA13, PPA14, PPA17, and PPA18 isolated from cucumber, tomato, eggplant, and chili exhibited higher virulence and pathogenicity. Correlation studies indicated that nearly 40% of mortality in C. elegans was triggered by the P. aeruginosa strains with high levels of pyocyanin (> 9 µg/ml) and biofilm to planktonic ratio (> 8). Conclusion This study demonstrated that plant-associated P. aeruginosa could be a potential threat to human health similar to the clinical strains. Pyocyanin could be a potential biomarker to screen the pathogenic P. aeruginosa strains in the agricultural ecosystem.


Background
Pseudomonas aeruginosa is an omnipresent bacterium commonly found in soil, water, moist surfaces, plants, animals, and humans. This bacterium is an opportunistic pathogen that causes terminal infections in patients with a weakened immune system. Its secondary metabolites, pyocyanin, rhamnolipid, and siderophores (pyochelin and pyoverdine), play a major role in establishing human infections [1]. In addition, biofilm formation is the key factor for P. aeruginosa-associated chronic obstructive lung infections [2]. Unfortunately, agricultural soil and plants have been the vast reservoirs of this bacterium [3]. Most of the P. aeruginosa strains in the agricultural systems are helpful for plant growth and protection [4][5][6]. However, some have also caused wilt and rot in the host plants [7,8]. Despite multiple reports on the prevalence of P. aeruginosa in agricultural systems, there are limited studies on determining the associated health risks. Considering the opportunistic pathogenicity of this bacterium, it is crucial to examine the bio-safety of plant-associated P. aeruginosa. Caenorhabditis elegans is the standard model system used to determine the pathogenicity of clinical P. aeruginosa strains [9]. It is a bacterivorous nematode that can be cultured in the laboratory by supplying Escherichia coli OP50 as the food source. The C. elegans worms imitate the human innate immunity and rapidly exhibit sickness and death when fed virulent bacterial cells [10]. This model system can be used to determine if the ecological adaptation has reduced the virulence and pathogenicity of plant-associated P. aeruginosa. This might either validate the possible use of the plant-associated P. aeruginosa strains for agricultural sustainability or reveal their pathogenicity level and associated health hazards.
In our previous study, we isolated and characterized P. aeruginosa strains (PPA01-PPA18) from rhizospheric and endophytic niches of cucumber, tomato, eggplant, and chili harvested from different farms [11]. These strains had plant-beneficial traits such as mineral solubilization and plant-growth hormone production. In addition, all the strains inhibited the growth of bacterial and fungal phytopathogens, including Xanthomonas oryzae, Pythium aphanidermatum, Rhizactonia solani, and Fusarium oxysporum [12]. However, these plant-associated strains had several virulence traits, such as the production of pyocyanin, rhamnolipid, and siderophore, biofilm formation, swarming motility, and multiple lytic activities [11,12]. In the current work, we hypothesized that one of these virulence factors could be used as a biomarker to detect the pathogenicity level of P. aeruginosa in agricultural systems. We tested if the variations in virulence factors can alter the impact of plant-associated P. aeruginosa on C. elegans survival. We have attempted to identify the critical virulence factor(s) contributing to the pathogenicity through this approach.

C. elegans survival on plant-associated Pseudomonas aeruginosa strains
The survival of C. elegans worms on feeding the plantassociated P. aeruginosa strains was predicted based on death, paralysis, and egg-laying ( Fig. 1). Overall, three clinical P. aeruginosa strains (positive controls) caused significantly higher mortality (47-100%) when compared with the agricultural strains (12-42%) ( Fig. 1A and B). The reproductive health of the nematodes also drastically declined on feeding these strains (Fig. 1C). The nematodes fed with E. coli OP50 (negative control) exhibited the least death and paralysis, and their egg-laying ability remained unaffected (Fig. 1). All of the plant-associated P. aeruginosa strains caused a significantly higher negative impact on the nematode health when compared with the negative control (OP50). PPA03/cucumber; PPA08, PPA10/tomato; PPA13 and PPA14/eggplant; PPA17 and PPA18/chili were identified as the most pathogenic ones.

Relative analyses of virulence factors and C. elegans pathogenicity in plant-associated P. aeruginosa strains
Principal component analysis (PCA) identified the relation between plant-associated P. aeruginosa strains (PPA01-PPA18), their virulence factors (pyocyanin, rhamnolipid, siderophore, and biofilm), and C. elegans reproductive (egg-laying) and survival (paralysis and death) traits. The PCA biplot with two principal components (Dim1 and Dim2) depicted the orthogonal positions of the P. aeruginosa strains along with their virulence factors and pathogenicity against C. elegans ( Fig. 2A). Dim1 and Dim2 contributed to 72.5% and 9% variability, respectively (Fig. 2B). Among the tested variables, the number of eggs laid by the C. elegans and paralytic and dead worms in 24, 48, and 72 h had high loading values (> 7.5) and significantly contributed to the Dim1 (Fig. 2C)  negative quadrant also had a few PPA strains (PPA07/ tomato; PPA11, PPA13/eggplant; PPA17/chili).

Segregating the plant-associated P. aeruginosa strains based on their pathogenicity against C. elegans
K-means clustering analyses grouped the P. aeruginosa strains based on their impact on the reproduction and survival of the C. elegans nematodes (Fig. 3). Cluster A had the E. coli OP50 (negative control) along with 11 plant-associated P. aeruginosa strains (PPA01, PPA02, PPA04/cucumber; PPA05, PPA06, PPA07, PPA09/ tomato; PPA11/eggplant; PPA15, PPA16/chili) that had low impact on the C. elegans survivability. The heatmap indicated that these strains had low virulence factors and led to minimal death and paralysis in C. elegans. The nematodes feeding on these strains laid a comparatively high number of eggs after 24, 48, and 72 h. Cluster B was occupied by the highly virulent clinical isolates of P.
aeruginosa (PAO1, ATCC10145, and ATCC9027) along with eight plant-associated P. aeruginosa strains (PPA03/ cucumber; PPA08, PPA10/tomato; PPA13, PPA14/eggplant; PPA17, PPA18/chili) that significantly affected the reproduction and survival of the C. elegans. These strains drastically reduced the number of eggs laid by the C. elegans (indicated by yellow) and led to a high number of paralytic-and dead worms (indicated by blue) after 24, 48, and 72 h of feeding. All the tested variables were clustered into two groups. The P. aeruginosa virulence factors and the number of paralytic-and dead worms clustered together. The number of eggs laid by the C. elegans from 24 to 72 h of feeding was grouped separately.

Correlation between P. aeruginosa virulence factors and C. elegans survivability
Pearson correlation coefficient analyses determined the impact of each virulence factor produced by the plant-associated P. aeruginosa on the survivability of the C. elegans model (Fig. 4). The correlogram was created with a scale of -1 to 1 (red to blue). Among the four virulence traits tested, pyocyanin production and biofilm formation had the highest impact (> 0.65) on paralysis and death in C. elegans worms. The rhamnolipid had a moderate impact (> 0.5) on the C. elgans survival, while the siderophore exhibited the most negligible impact (< 0.25). The P. aeruginosa strains that produced high levels of pyocyanin (PPA14/eggplant; PPA18/chili) or biofilm (PPA03/cucumber; PPA10/tomato) caused high mortality   Fig. 5).

Discussion
Pseudomonas aeruginosa is an opportunistic human pathogen that is omnipresent in multiple ecosystems. The strains that thrive in the agricultural system efficiently promote plant growth and inhibit phytopathogens [13,14]. However, this bacterium is a Priority Level-I critical pathogen that causes terminal infections in immune-compromised individuals [15]. So far, there have been minimal works on the biosafety of plant-associated P. aeruginosa strains [16]. Our previous studies identified that agricultural P. aeruginosa strains harboring plantbeneficial traits could also exhibit virulence and pathogenicity [11,12]. The present work identified the critical virulence factors that contribute to the eukaryotic pathogenesis of plant-associated P. aeruginosa using the C. elegans model system. C. elegans is a standard model system used to determine the ability of clinical P. aeruginosa strains to cause mammalian infections [9,17]. Several studies have demonstrated the pathogenicity of clinical P. aeruginosa strains using the C. elegans model [18][19][20]. However, the present work is the first attempt to use the C. elegans model to test the biosafety of plant-associated P. aeruginosa. C. elegans slow-killing is usually triggered due to the accumulation of pathogenic P. aeruginosa cells within the intestinal lumen of the nematodes [9]. In our study, the slow-killing assay revealed that plant-associated P. aeruginosa strains, PPA01-PPA18, exhibited relatively lower pathogenicity against C. elegans when compared to the clinical isolates, ATCC10145, ATCC9047, and PAO1. However, the most pathogenic PPA strains from eggplant (PPA14) and chili (PPA18) caused up to 43% of worm mortality.
Notably, these two strains produced more than 9 µg/ ml of pyocyanin under in vitro conditions. Pyocyanin is a unique secondary metabolite produced by P. aeruginosa. It is a redox-active zwitterion that rapidly generates reactive oxygen species, leading to organ damage in the eukaryotic hosts [1,21]. P. aeruginosa uses its pyocyanin to cause cytotoxic effects on respiratory, urological, vascular, and central nervous systems leading to multiple organ damage in the eukaryotic host [22]. A study on a highly pathogenic clinical strain, P. aeruginosa PA14, showed that three phenazine compounds such as 1-hydroxyphenazine, phenazine-1-carboxylic acid, and pyocyanin contribute to its pathogenicity [23]. In the current work, the plant-associated P. aeruginosa strains that produced high pyocyanin levels significantly affected C. elegans reproduction and survival (Fig. 5). Biofilm-forming P. aeruginosa causes chronic infections and increases the mortality rate in patients with critical pulmonary conditions [24][25][26]. The biofilm shields the P. aeruginosa cells from the host immune system and antibiotics, thereby facilitating persistent colonization [27]. In this study, we observed that high biofilm-formers could easily trigger C. elegans mortality. The plant-associated P. aeruginosa strains, PPA03 and PPA10, isolated from cucumber and tomato plants, respectively, had high biofilm levels and caused 30% death of the nematodes. Rhamnolipid is one of the key virulence factors of P. aeruginosa that rupture the epithelial cells enabling the infiltration of mammalian lung tissues [28]. However, high rhamnolipid producing P. aeruginosa strains (PPA01/cucumber; PPA16/chili) caused relatively low death compared to the high pyocyanin producers. In the k-means clustering analyses, these strains occupied the low virulence group. Similarly, the siderophore production was less correlated with C. elegans paralysis and mortality than pyocyanin, rhamnolipid, and biofilm. However, pyoverdine-mediated hypoxia and death have previously been reported in the C. elegans fed with clinical P. aeruginosa [18].
Overall, seven out of these eighteen PPA strains (PPA03/cucumber; PPA08, and PPA10/tomato; PPA13, and PPA14/eggplant; PPA17, and PPA18/chili) tested in this study clustered together with the clinical strains based on their virulence and C. elegans pathogenicity (Fig. 2). This shows that the non-clinical P. aeruginosa strain could also be hazardous to human and animal health. Our study identified pyocyanin production and biofilm formation as the major pathogenicity determinants in the plant-associated P. aeruginosa. These two factors could be used as biomarkers to segregate the virulent and avirulent P. aeruginosa strains in the agricultural ecosystem.

Conclusion
In conclusion, the plant-associated P. aeruginosa strains showed wide variation in their virulence factors which in turn alters their pathogenicity levels. Despite expressing comparatively lesser virulence than the clinical isolates, the plant-associated P. aeruginosa strains are pathogenic enough to cause paralysis and mortality in the C. elegans model. The P. aeruginosa strains in the agricultural ecosystem might evolve more pathogenic when exposed to the human and animal environment. Numerous studies have recommended P. aeruginosa strains to promote plant growth, alleviate abiotic stress, and protect plants against pests and insects [5,8,29,30]. Based on our results, biosafety assessment is crucial before recommending an opportunistic bacterium for plant growth and protection. The risk associated with the agriculturally important P. aeruginosa strains can be detected based on their pyocyanin and biofilm levels. Such virulent and pathogenic P. aeruginosa strains in edible plants could cause potential health hazards to plants, animals, and humans with a weakened immune system.

Strains and culture conditions
Plant-associated P. aeruginosa strains used in this study were previously isolated by the authors from cucumber, tomato, eggplant, and chili (Table 2; [11]). Clinical P. aeruginosa strains, PAO1, ATCC10145, and ATCC9027, were used as positive controls for pathogenicity assays [31][32][33]. These strains were grown at 37 °C in the Pseudomonas agar (for pyocyanin) medium (Himedia). C. elegans N2 hermaphrodite strain was cultured at 20 °C in the nematode growth medium (NGM) overlaid with Escherichia coli strain OP50 as a food source. The E. coli OP50 was periodically sub-cultured in the Luria Bertani (LB) medium and was used as a negative control for C. elegans reproduction and survival assays in all the experiments.

Biofilm estimation
The P. aeruginosa cultures were grown in LB broth for 72 h, and the biofilm formation was estimated using the standard crystal violet-microtitre assay [34]. In brief, 25 µl of 24 h old cultures of the P. aeruginosa strains (OD660 ~ 0.5) were inoculated into 225 μl of LB broth in microtitre wells. After 72 h of incubation, A660 was measured (Spectramax ® i3x, USA) to estimate the planktonic population. Biofilm attached to the microtitre wells was washed with sterile water and drenched with 300 µl of 0.1% crystal violet. After 10-15 min of incubation at room temperature, the plate was delicately washed with sterile water and allowed to dry for 24 h at room temperature. After 24 h, biofilm was dissolved using 30% acetic acid (300 µl), and absorbance was measured at 550 nm. Biofilm to the planktonic ratio (B:P) was determined for all the tested strains.

Pyocyanin estimation
For pyocyanin assay, the cultures were grown in glycinealanine broth for 48 h [35]. The pyocyanin was extracted from the cell-free supernatant using chloroform and spectrophotometrically quantified at 520 nm [36]. The A 520 was multiplied with the pyocyanin extinction coefficient (17.072) to determine the concentration (µg/ml) [37].

Rhamnolipid estimation
Rhamnolipid production was induced by growing the cells in protease peptone ammonium salts broth with a 2% (v/v) sunflower oil supplement [38]. Crude rhamnolipid was separated from the cell-free supernatant by chloroform-methanol extraction and quantified using the gravimetric method [39]. Briefly, the cell-free supernatant of 7 days old cultures was acidified with 12 M hydrochloric acid, and the rhamnolipid was extracted using a chloroform-methanol (2:1) mixture. The extracted lipids were concentrated, weighed, and expressed as µg/ml of the culture supernatant.

Siderophore estimation
P. aeruginosa strains were grown overnight in succinate broth [40], and Chrome Azurol S (CAS)-shuttle assay was performed to quantify the total siderophore [41]. Briefly, an equal volume of CAS solution was added to the cell-free supernatant and incubated for an hour at ambient temperature. The absorbance was measured at 630 nm, and the percentage of siderophore was estimated based on the equation [(A r -A s )/A r ] × 100, where A r refers to the A 630 of reference solution (mixture of CAS solution and uninoculated broth) and A s refers to the A 630 of the sample (mixture of CAS solution and culture supernatant) [40].

C. elegans reproduction and survival assay
C. elegans gravid adults were ruptured using 1 N NaOH and 5% sodium hypochlorite (1:1) solution [42]. Their eggs were incubated in an M9 buffer for 24 h to allow hatching. L1-nematodes that emerged from these eggs were released into fresh OP50 lawns on NGM plates and allowed to grow up to the L4 stage. These L4-worms (20 per plate) were then released on NGM seeded with 50 µl of overnight grown P. aeruginosa strains (OD660 ~ 0.5) and were incubated at 20 °C [9,10]. The reproductive ability of these worms was constantly monitored based on the number of eggs laid after 24, 48, and 72 h of feeding. The impact of P. aeruginosa strains on C. elegans survival was estimated based on paralysis and death. The worms were scored paralytic when they turned non-motile postfeeding. The nematodes that did not respond to physical stimulus were considered dead. The paralytic and dead worms were counted at 24, 48, and 72 h of incubation. All the experiments were performed with three replicates.

Statistical analyses
The statistical analyses were performed in R software (Version 4.1.1) (R Core Team, Vienna, Austria). The C. elegans pathogenicity data were tested with a one-way analysis of variance (ANOVA) followed by Tukey's honestly significant difference test at α = 0.05. The PCA was performed for all the assessed variables using the princomp function of the factoextra-package of R. The PCA biplot, contribution plot, and eigenvalues corresponding to the variation explained by each principal component were visualized using the fviz function of factoextra. K-means clustering heatmap was generated using the heatmap.2 R package to group the PPA strains based on their virulence factors and C. elegans survival. The correlation between the assessed variables was evaluated based on Pearson's correlation and visualized through Corrplot-package.