Effect of Phthalates and Their Substitutes on the Physiology of Pseudomonas aeruginosa

Abstract

Phthalates are used in a variety of applications-for example, as plasticizers in polyvinylchloride products to improve their flexibility-and can be easily released into the environment. In addition to being major persistent organic environmental pollutants, some phthalates are responsible for the carcinogenicity, teratogenicity, and endocrine disruption that are notably affecting steroidogenesis in mammals. Numerous studies have thus focused on deciphering their effects on mammals and eukaryotic cells. While multicellular organisms such as humans are known to display various microbiota, including all of the microorganisms that may be commensal, symbiotic, or pathogenic, few studies have aimed at investigating the relationships between phthalates and bacteria, notably regarding their effects on opportunistic pathogens and the severity of the associated pathologies. Herein, the effects of phthalates and their substitutes were investigated on the human pathogen, Pseudomonas aeruginosa, in terms of physiology, virulence, susceptibility to antibiotics, and ability to form biofilms. We show in particular that most of these compounds increased biofilm formation, while some of them enhanced the bacterial membrane fluidity and altered the bacterial morphology.

Keywords: Pseudomonas aeruginosa; antibiotic susceptibility; biofilm; phthalates; virulence.

Figure 1

Phthalates and substitutes used in this study. High molecular weight phthalates: DEHP (2-éthylhexyl)-phthalate), DINP (bis (7-methyloctyl) phthalate). Low molecular weight phthalates: DEP (diethyl phthalate), and DBP (dibutyl phthalate). Substitutes: DEHT (bis (2-ethylhexyl) terephthalate), ATBC (tributyl acetylcitrate), and TXIB (2,2,4-trimethyl-1,3-pentanediol diisobutyrate). Other substitutes: DOIP (di-2-ethylhexyl isophthalate) and DIOP (dicyclohexylphthalate).

Figure 2

Phthalates or substitutes did not affect P. aeruginosa growth. Compounds were added at concentrations ranging from 10⁻¹⁰ to 10⁻³ M to the growth medium prior to P. aeruginosa inoculation. Growth was monitored for 24 h. GC: growth control in M9 containing succinate as sole carbon source without any phthalates or substitutes.

Figure 3

Phthalates or substitutes did not alter pyocyanin production. Phthalate and substitute concentrations ranging from 10⁻³ to 10⁻¹⁰ M (colors indicated in the scale). GC: P. aeruginosa’s growth control in M9 succinate medium without phthalates or substitutes. NS = p > 0.05.

Figure 4

Effect of phthalates and substitutes on pyoverdine production when P. aeruginosa was grown in microtiter plates (A) or in Erlenmeyer flasks (B). Phthalate and substitute concentrations ranging from 10⁻³ to 10⁻¹⁰ M (colors indicated in the scale). GC: P. aeruginosa’s growth control inM9 succinate medium without any phthalates or substitutes. NS = p > 0.05; * = p < 0.05; ** = p < 0.01; *** = p < 0.001.

Figure 5

Phthalates and substitutes affect biofilm formation. Biofilms were investigated in polystyrene plates (A), at the air–liquid interface (B), by confocal laser scanning microscope observation (C), and by COMSTAT analysis of their biovolumes (D). NS = p > 0.05; * = p < 0.05; ** = p < 0.01; *** = p < 0.001.

Figure 6

Effect of phthalates and substitutes on membrane fluidity. Anisotropy index variations were observed upon addition of phthalates or substitutes at 10⁻³ M to P. aeruginosa. Graphs represent means ± SEM. NS = p > 0.05; *** = p < 0.001, **** = p < 0.0001.

Figure 7

Some phthalates can alter the morphology of P. aeruginosa. Bacteria were observed by scanning electron microscopy after being grown for 24 h in M9 succinate medium under agitation in the presence of phthalates or substitutes at 10⁻³ M. The micrographs presented are representative of 10 images taken for each sample obtained from a biological duplicate. The white arrows indicate the presence of holes in the bacterial envelope. The magnification is ×20,000 and the bars represent 4 µm.