Skip to main content
Download PDF

Pathogenesis of plant-associated Pseudomonas aeruginosa in Caenorhabditis elegans model

BMC Microbiology volume 22, Article number: 269 (2022) Cite this article

Abstract

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.

Peer Review reports

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.

Results

C. elegans survival on plant-associated Pseudomonas aeruginosa strains

The survival of C. elegans worms on feeding the plant-associated 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.

Fig. 1
figure 1

A Death, (B) paralysis, and (C) egg count of C. elegans as influenced by plant-associated and clinical strains of P. aeruginosa. ATCC10145, ATCC9027, and PAO1 – clinical strains; PPA01 to PPA18 – plant-associated strains; Std(OP50)—E. coli (OP50) standard nematode-feeding strain. Data represent the mean values (n = 3), and error bars indicate the standard error. For each panel, different letters indicate significant differences among the strains according to Tukey's test (α < 0.05)

Full size image

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). The P. aeruginosa virulence factors majorly contributed to the Dim2. The E. coli OP50 (negative control) was positioned in the negative quadrant of the PCA plot while the clinical P. aeruginosa strains (positive controls) occupied the positive quadrant. Four plant-associated P. aeruginosa strains (PPA03/cucumber; PPA10/tomato; PPA14/eggplant; PPA18/chili) occupied the positive quadrant. The negative quadrant also had a few PPA strains (PPA07/tomato; PPA11, PPA13/eggplant; PPA17/chili).

Fig. 2
figure 2

PCA relating virulence factors of plant-associated P. aeruginosa strains and their pathogenicity against C. elegans. A PCA biplot showing the position of each strain along with the orthogonal positions of the observed variables. The percentage variance explained by each principal component (Dim1 and Dim2) is given in parentheses in axes. B The percent contribution of each principal component to the cumulative variability in PCA. C The percent contribution of each variable on the axis identified by the principal component analysis. The red dotted line indicates significant loading values (> 0.70). D – death; P – paralysis; E – eggs at 24, 48, and 72 h incubation. ATCC10145, ATCC9027, and PAO1 – clinical strains; PPA01 to PPA18 – plant-associated strains; OP50—E. coli (OP50) standard nematode-feeding strain

Full size image

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.

Fig. 3
figure 3

K-means clustering analysis of P. aeruginosa strains based on their pathogenicity against C. elegans. Double dendrogram and heatmap were created based on the K-means clustering and Spearman distance methods. The heatmap indicates the virulence and pathogenicity of the P. aeruginosa strains (yellow to blue – negative to positive). The top dendrogram reflects the clustering of the observed variables, while the left dendrogram indicates the grouping of the P. aeruginosa strains. D – death; P – paralysis; E – eggs at 24, 48, and 72 h incubation

Full size image

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).

Fig. 4
figure 4

Pearson correlation plot for the P. aeruginosa virulence factors and C. elegans survival. Blue indicates positive correlation, and red indicates negative correlation. The level of correlation was further visualized through ellipses and their angles. D – death; P – paralysis; E – eggs at 24, 48, and 72 h incubation

Full size image

The P. aeruginosa strains that produced high levels of pyocyanin (PPA14/eggplant; PPA18/chili) or biofilm (PPA03/cucumber; PPA10/tomato) caused high mortality and paralysis in C. elegans regardless of low rhamnolipid levels (Table 1; Fig. 5). On the contrary, the P. aeruginosa strains (PPA01/cucumber; PPA16/chili) with high rhamnolipid levels but less pyocyanin and biofilm caused relatively low paralysis and mortality in C. elegans. The clinical isolate, P. aeruginosa PAO1, which expressed high levels of all three virulence factors, triggered severe paralysis and death of the nematodes, while the E. coli OP50 (negative control) did not cause any harmful impact (Table 1; Fig. 5).

Table 1 Comparing the virulence levels and C. elegans pathogenicity of select strains of plant-associated P. aeruginosa
Full size table
Fig. 5
figure 5

Impact of select strains of plant-associated P. aeruginosa on C. elegans. A Egg-laying, B Paralysis, and C Mortality. OP50 – negative control; P. aeruginosa PPA01/cucumber and PPA16/chili – high rhamnolipid producers with low levels of biofilm and pyocyanin; PPA03/cucumber and PPA10/tomato – high biofilm formers with low rhamnolipid and pyocyanin levels; PPA14/eggplant and PPA18/chili – high pyocyanin producers but low rhamnolipid and biofilm

Full size image

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 plant-beneficial 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.

Materials and methods

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.

Table 2 Bacteria strains used in this study
Full size table

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 glycine-alanine broth for 48 h [35]. The pyocyanin was extracted from the cell-free supernatant using chloroform and spectrophotometrically quantified at 520 nm [36]. The A520 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 [(Ar—As)/Ar] × 100, where Ar refers to the A630 of reference solution (mixture of CAS solution and uninoculated broth) and As refers to the A630 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 post-feeding. 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.

Availability of data and materials

The datasets generated and/or analyzed during the current study are available from the corresponding author on reasonable request.

Abbreviations

PPA:

Plant-associated Pseudomonas aeruginosa

PCA:

Principal component analysis

DIM:

Dimension

LB:

Luria Bertani

B:P:

Biofilm:Planktonic

CAS:

Chrome-Azural S

ANOVA:

Analysis of variance

References

  1. Moradali MF, Ghods S, Rehm BHA. Pseudomonas aeruginosa lifestyle: A paradigm for adaptation, survival, and persistence. Front Cell Infect Microbiol. 2017;7(39):1–29.

    Google Scholar 

  2. Mulcahy LR, Isabella VM, Lewis K. Pseudomonas aeruginosa biofilms in disease. Microb Ecol. 2014;68(1):1–12.

    Article  CAS  PubMed  Google Scholar 

  3. Green SK, Schroth MN, Cho JJ, Kominos SD, Vitanza-Jack VB. Agricultural plants and soil as a reservoir for Pseudomonas aeruginosa. Appl Microbiol. 1974;28(6):987–91.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Gupta V, Buch A. Pseudomonas aeruginosa predominates as multifaceted rhizospheric bacteria with combined abilities of P-solubilization and biocontrol. J Pure Appl Microbiol. 2019;13(1):319–28.

    Article  CAS  Google Scholar 

  5. Sancheti A, Ju L-K. Eco-friendly rhamnolipid based fungicides for protection of soybeans from Phytophthora sojae. Pest Manag Sci. 2019;75(11):3031–8.

    Article  CAS  PubMed  Google Scholar 

  6. Chandra H, Kumari P, Bisht R, Prasad R, Yadav S. Plant growth promoting Pseudomonas aeruginosa from Valeriana Wallichii displays antagonistic potential against three phytopathogenic fungi. Mol Biol Rep. 2020;47(8):6015–26.

    Article  CAS  PubMed  Google Scholar 

  7. Gao J, Wang Y, Wang CW, Lu BH. First report of bacterial root rot of ginseng caused by Pseudomonas aeruginosa in China. Plant Dis. 2014;98(11):1577–1577.

    Article  CAS  PubMed  Google Scholar 

  8. Tiwari P, Singh JS. A plant growth promoting rhizospheric Pseudomonas aeruginosa strain inhibits seed germination in Triticum aestivum (L) and Zea mays (L). Microbiol Res. 2017;8(2):7233.

    Article  Google Scholar 

  9. Tan M-W, Mahajan-Miklos S, Ausubel FM. Killing of Caenorhabditis elegans by Pseudomonas aeruginosa used to model mammalian bacterial pathogenesis. PNAS. 1999;96(2):715–20.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Adonizio A, Kong K-F, Mathee K. Inhibition of quorum sensing-controlled virulence factor production in Pseudomonas aeruginosa by South Florida plant extracts. Antimicrob Agents Chemother. 2008;52(1):198–203.

    Article  CAS  PubMed  Google Scholar 

  11. Ambreetha S, Marimuthu P, Mathee K, Balachandar D. Rhizospheric and endophytic Pseudomonas aeruginosa in edible vegetable plants share molecular and metabolic traits with clinical isolates. J Appl Microbiol. 2021;132(4):3226–48.

    Article  PubMed  Google Scholar 

  12. Ambreetha S, Marimuthu P, Mathee K, Balachandar D. Plant-associated Pseudomonas aeruginosa strains harbor multiple virulence traits critical for human infection. J Med Microbiol. 2022;71(8):1–18.

    Article  Google Scholar 

  13. Ahemad M, Khan MS. Phosphate-solubilizing and plant-growth-promoting Pseudomonas aeruginosa PS1 improves greengram performance in quizalafop-p-ethyl and clodinafop amended soil. Arch Environ Contam Toxicol. 2010;58(2):361–72.

    Article  CAS  PubMed  Google Scholar 

  14. Sun X, Xu Y, Chen L, Jin X, Ni H. The salt-tolerant phenazine-1-carboxamide-producing bacterium Pseudomonas aeruginosa NF011 isolated from wheat rhizosphere soil in dry farmland with antagonism against Fusarium graminearum. Microbiol Res. 2021;245: 126673.

    Article  CAS  PubMed  Google Scholar 

  15. TalebiBezminAbadi A, Rizvanov AA, Haertlé T, Blatt NL. World Health Organization report: Current crisis of antibiotic resistance. BioNanoScience. 2019;9(4):778–88.

    Article  Google Scholar 

  16. Kumar A, Munder A, Aravind R, Eapen SJ, Tümmler B, Raaijmakers JM. Friend or foe: genetic and functional characterization of plant endophytic Pseudomonas aeruginosa. Environ Microbiol. 2013;15(3):764–79.

    Article  CAS  PubMed  Google Scholar 

  17. Tan MW, Rahme LG, Sternberg JA, Tompkins RG, Ausubel FM. Pseudomonas aeruginosa killing of Caenorhabditis elegans used to identify P. aeruginosa virulence factors. PNAS. 1999;96(5):2408–13.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Kirienko NV, Cezairliyan BO, Ausubel FM, Powell JR. Pseudomonas aeruginosa PA14 pathogenesis in Caenorhabditis elegans. In: Filloux A, Ramos JL, editors. Pseudomonas Methods and Protocols. New York, NY: Springer New York; 2014. p. 653–69.

    Chapter  Google Scholar 

  19. Kirienko NV, Kirienko DR, Larkins-Ford J, Wählby C, Ruvkun G, Ausubel FM. Pseudomonas aeruginosa disrupts Caenorhabditis elegans iron homeostasis, causing a hypoxic response and death. Cell Host Microbe. 2013;13(4):406–16.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Gallagher LA, Manoil C. Pseudomonas aeruginosa PAO1 kills Caenorhabditis elegans by cyanide poisoning. J Bacteriol. 2001;183(21):6207–14.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Moayedi A, Nowroozi J, Akhavan Sepahy A. Cytotoxic effect of pyocyanin on human pancreatic cancer cell line (Panc-1). Iran J Basic Med Sci. 2018;21(8):794–9.

    PubMed  PubMed Central  Google Scholar 

  22. Hall S, McDermott C, Anoopkumar-Dukie S, McFarland AJ, Forbes A, Perkins AV, Davey AK, Chess-Williams R, Kiefel MJ, Arora D, et al. Cellular effects of pyocyanin, a secreted virulence factor of Pseudomonas aeruginosa. Toxins. 2016;8(8):236–44.

    Article  PubMed Central  Google Scholar 

  23. Cezairliyan B, Vinayavekhin N, Grenfell-Lee D, Yuen GJ, Saghatelian A, Ausubel FM. Identification of Pseudomonas aeruginosa phenazines that kill Caenorhabditis elegans. PLoS Pathog. 2013;9(1): e1003101.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Nixon GM, Armstrong DS, Carzino R, Carlin JB, Olinsky A, Robertson CF, Grimwood K. Clinical outcome after early Pseudomonas aeruginosa infection in cystic fibrosis. J Pediatr. 2001;138(5):699–704.

    Article  CAS  PubMed  Google Scholar 

  25. Bjarnsholt T, Jensen PO, Fiandaca MJ, Pedersen J, Hansen CR, Andersen CB, Pressler T, Givskov M, Høiby N. Pseudomonas aeruginosa biofilms in the respiratory tract of cystic fibrosis patients. Pediatr Pulmonol. 2009;44(6):547–58.

    Article  PubMed  Google Scholar 

  26. Singh PK, Schaefer AL, Parsek MR, Moninger TO, Welsh MJ, Greenberg EP. Quorum-sensing signals indicate that cystic fibrosis lungs are infected with bacterial biofilms. Nature. 2000;407(6805):762–4.

    Article  CAS  PubMed  Google Scholar 

  27. Abdulhaq N, Nawaz Z, Zahoor MA, Siddique AB. Association of biofilm formation with multi drug resistance in clinical isolates of Pseudomonas aeruginosa. EXCLI J. 2020;19:201–8.

    PubMed  PubMed Central  Google Scholar 

  28. Zulianello L, Canard C, Köhler T, Caille D, Lacroix J-S, Meda P. Rhamnolipids are virulence factors that promote early infiltration of primary human airway epithelia by Pseudomonas aeruginosa. Infect Immun. 2006;74(6):3134–47.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Roychowdhury R, Qaiser TF, Mukherjee P, Roy M. Isolation and characterization of a Pseudomonas aeruginosa strain PGP for plant growth promotion. Proc Natl Acad Sci India, Sect B Biol Sci. 2019;89(1):353–60.

    Article  CAS  Google Scholar 

  30. Monnier N, Furlan A, Botcazon C, Dahi A, Mongelard G, Cordelier S, Clément C, Dorey S, Sarazin C, Rippa S. Rhamnolipids from Pseudomonas aeruginosa are elicitors triggering Brassica napus protection against Botrytis cinerea without physiological disorders. Front Plant Sci. 2018;9:1170.

  31. Holloway BW. Genetic recombination in Pseudomonas aeruginosa. J Gen Microbiol. 1955;13(3):572–81.

    CAS  PubMed  Google Scholar 

  32. Haynes WC. Pseudomonas aeruginosa–-its characterization and identification. Microbiol. 1951;5(5):939–50.

    CAS  Google Scholar 

  33. Picard B, Denamur E, Barakat A, Elion J, Goullet P. Genetic heterogeneity of Pseudomonas aeruginosa clinical isolates revealed by esterase electrophoretic polymorphism and restriction fragment length polymorphism of the ribosomal RNA gene region. J Med Microbiol. 1994;40(5):313–22.

    Article  CAS  PubMed  Google Scholar 

  34. O’Toole GA. Microtiter dish biofilm formation assay. J Viz Exp. 2011;47: e2437.

    Google Scholar 

  35. Ingledew WM, Campbell JJ. A new resuspension medium for pyocyanine production. Can J Microbiol. 1969;15(6):595–8.

    Article  CAS  PubMed  Google Scholar 

  36. Essar DW, Eberly L, Hadero A, Crawford IP. Identification and characterization of genes for a second anthranilate synthase in Pseudomonas aeruginosa: interchangeability of the two anthranilate synthases and evolutionary implications. J Bacteriol. 1990;172(2):884–900.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Kurachi M. Studies on the biosynthesis of pyocyanine (II): Isolation and determination of pyocyanine. Bulletin of the Institute for Chemical Research, Kyoto University. 1958;36(6):174–87.

    Google Scholar 

  38. Zhang Y, Miller RM. Enhanced octadecane dispersion and biodegradation by a Pseudomonas rhamnolipid surfactant (biosurfactant). Appl Environ Microbiol. 1992;58(10):3276–82.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Gunther NW, Nunez A, Fett W, Solaiman DK. Production of rhamnolipids by Pseudomonas chlororaphis, a nonpathogenic bacterium. Appl Environ Microbiol. 2005;71(5):2288–93.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Pérez-Miranda S, Cabirol N, George-Téllez R, Zamudio-Rivera LS, Fernández FJ. O-CAS, a fast and universal method for siderophore detection. J Microbiol Methods. 2007;70(1):127–31.

    Article  PubMed  Google Scholar 

  41. Schwyn B, Neilands JB. Universal chemical assay for the detection and determination of siderophores. Anal Biochem. 1987;160(1):47–56.

    Article  CAS  PubMed  Google Scholar 

  42. Brenner S. The genetics of Caenorhabditis elegans. Genetics. 1974;77(1):71–94.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

S Ambreetha acknowledges the Fulbright Doctoral Nehru Research Fellowship provided by the US Department of State's Bureau of Educational and Cultural Affairs and the United-States India Educational Foundation (ID. PS00299273). We thank Dr. Kavita Babu (Center for Neuroscience, Indian Institute of Science, Bangalore, India) for kindly supplying the C. elegans nematodes and Dr. Sriyutha Murthy (Indira Gandhi Centre for Atomic Research, Kalpakkam, Tamilnadu, India) for providing P. aeruginosa PAO1.

Funding

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Author information

Authors and Affiliations

  1. Department of Agricultural Microbiology, Tamil Nadu Agricultural University, Coimbatore, 641003, Tamil Nadu, India

    Sakthivel Ambreetha & Dananjeyan Balachandar

Authors
  1. Sakthivel Ambreetha
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Dananjeyan Balachandar
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

DB framed the outline of the investigation. SA executed the experiments and processed the data. SA and DB wrote the manuscript. All authors reviewed and approved the manuscript.

Corresponding author

Correspondence to Dananjeyan Balachandar.

Ethics declarations

Ethics approval and consent to participate

All methods followed in this work comply with the guidelines and regulations. This work does not involve field studies on plants. The plant materials were collected for the isolation of Pseudomonas aeruginosa strains as per the institutional guidelines. No animal or human subject was used in this study.

Consent for publication

Not Applicable.

Competing interests

The authors declare no conflict of interests.

Additional information

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Ambreetha, S., Balachandar, D. Pathogenesis of plant-associated Pseudomonas aeruginosa in Caenorhabditis elegans model. BMC Microbiol 22, 269 (2022). https://doi.org/10.1186/s12866-022-02682-z

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1186/s12866-022-02682-z

Keywords

BMC Microbiology

ISSN: 1471-2180

Contact us