Virulence profiles of some Pseudomonas aeruginosa clinical isolates and their association with the suppression of Candida growth in polymicrobial infections

Abstract

Pseudomonas aeruginosa is an opportunistic pathogen that can cause a variety of diseases especially in the hospital environment. However, this pathogen also exhibits antimicrobial activity against Gram-positive bacteria and fungi. This study aimed to characterize different virulence factors, secreted metabolites and to study their role in the suppression of Candida growth. Fifteen P. aeruginosa isolates were tested for their anticandidal activity against 3 different Candida spp. by the cross-streak method. The effect on hyphae production was tested microscopically using light and scanning electron microscopy (SEM). Polymerase chain reaction was used in the detection of some virulence genes. Lipopolysaccharide profile was performed using SDS-polyacrylamide gel stained with silver. Fatty acids were analyzed by GC-MS as methyl ester derivatives. It was found that 5 P. aeruginosa isolates inhibited all tested Candida spp. (50-100% inhibition), one isolate inhibited C. glabrata only and 3 isolates showed no activity against the tested Candida spp. The P. aeruginosa isolates inhibiting all Candida spp. were positive for all virulence genes. GC-Ms analysis revealed that isolates with high anticandidal activity showed spectra for several compounds, each known for their antifungal activity in comparison to those with low or no anticandidal activity. Hence, clinical isolates of P. aeruginosa showed Candida species-specific interactions by different means, giving rise to the importance of studying microbial interaction in polymicrobial infections and their contribution to causing disease.

Fig 1. Anticandidal activity of the tested P. aeruginosa against 6 Candida Spp.

A: showed complete inhibition by P₅ strain to 5 Candida strains and showed (50%) ++ inhibition of C. glabrata. B: showed variable activity of P_S2 strain against the tested Candida: no inhibition was observed against C. albicans and one C. krusei; 25% (+) inhibition against one C. krusei and the tested C. glabrata.

Fig 2

A: C. albicans forming germ tube after their incubation in human serum for 3 hrs. B: showed C. albicans with no observable germ tubes after their incubation in human serum containing P. aeruginosa culture cell free filtrate for 3 hrs.

Fig 3

A: shows amplicons for phenazines encoding genes. B: shows amplicons for different virulence genes of the tested P. aeruginosa.

Fig 4. LPS SDS-polyacrylamide gel profile stained by silver nitrate.

Lanes 1, 2, 3&4 (P₅, P₈, P₉ and P₁₆ isolates with anticandidal activity)_. Lane 5 and Lane 6 (P₁₁₁ and P_{118 isolates} with no anticandidal activity).

Fig 5. Mass spectrum of the extracted LPS obtained from P₈ isolate.

A: Propanoic acid, 2-mercapto, methyl ester, B: Undecanoic acid, 3- hydroxyl, methyl ester, C: 1H Indole, 5-methyl-2-phenyl, D: Quorum sensing quinolones E: Rhamnolipids.

Fig 6. Mass spectrum of the extracted LPS obtained from P₅ isolate.

A: Butanoic acid, 2- methyl, heptyl ester, B: Propanoic acid, 2-mercapto, methyl ester antifungal, C: Furfural or 3, 5 Dimethyl pyrazole, D: 3- isoquinolinamine, E: Quorum sensing quinolones, F: Rhamnolipids.

Fig 7. Mass spectrum of the extracted LPS obtained from P_S3 isolate.

A: Propanoic acid, 2-mercapto, methyl ester (antifungal), B: Undecanoic acid, 3- hydroxyl, methyl ester, C: Quorum sensing quinolones D: Rhamnolipids.

Fig 8. Mass spectrum of the extracted LPS obtained from P_SA2 isolate.

A: Quorum sensing quinolones B: Rhamnolipids.

Fig 9. Scanning electron micrograph showing P. aeruginosa adhered to C. albicans after incubating the 2 organisms together for 48 hrs.

A: Candida cells showed irregular cell wall and some disrupted cells with no observable hyphae and scattered P. aeruginosa cells. B: SEM graph showed candida cells with disorganized and disrupted cell wall after their incubation with P. aeruginosa culture cell free filtrate.