Abstract
Introduction: Plastic pollution represents a critical environmental challenge, leading to a growing interest in biological degradation strategies using insect larvae. However, the biosafety risks associated with the microbial shifts during polymer digestion remain poorly understood. This study aimed to evaluate the implications of polystyrene (PS) biodegradation by Zophobas atratus larvae on biosafety, specifically testing the hypothesis that the stress of polymer digestion alters the gut microbiome structure and influences the antibiotic resistance and virulence of the opportunistic pathogen Pseudomonas aeruginosa.
Objective: To evaluate the implications of PS biodegradation by Zophobas atratus larvae on biosafety, specifically testing the hypothesis that the stress of polymer digestion alters the gut microbiome structure and influences the antibiotic resistance and virulence of the opportunistic pathogen Pseudomonas aeruginosa.
Methods: Z. atratus larvae were reared on a PS monodiet or a control oatmeal diet. Gut microbiota composition was analyzed via culture-dependent methods. P. aeruginosa strains isolated from the control (PAW) and plastic-modified (PAM) groups underwent antibiotic susceptibility testing (disk diffusion). Pyoverdine production was quantified spectrophotometrically at 400 nm for standardized bacterial densities (0.5 and 1.0 McF). Virulence was assessed in vivo using a murine model of acute pneumonia (n=107). The median lethal dose (LD50), absolute lethal dose (LD100), and Odds Ratio (OR) for mortality were calculated. Histopathological examination of lung tissue was performed to assess tissue damage.
Results: The PS diet induced a distinct taxonomic shift, leading to a statistically significant depletion of symbiotic Lactobacillus spp. (p = 0.0384) and the expansion of opportunistic Enterobacteriaceae, alongside the stable persistence of Pseudomonas spp. The plastic-adapted P. aeruginosa (PAM) isolate retained wild-type susceptibility to anti-pseudomonal antibiotics (carbapenems, fluoroquinolones). However, it exhibited phenotypic changes, including hyperproduction of pyoverdine. Spectrophotometric analysis revealed that PAM produced 9.85±0.21 µmol/L of pyoverdine at 0.5 McF, which is 32.4% higher (p < 0.0001) than the PAW strain 7.44±0.15 µmol/L). This gap remained significant at 1.0 McF (23.53 vs. 20.91 µmol/L, p = 0.0028). Preliminary in vivo profiling determined the LD50 at 3.2×10⁷ CFU for PAM and 5.6×10⁷ CFU for the wild-type (PAW). Based on these values, a targeted dose of 10⁷ CFU was selected for the main comparative assay. At this dose, the modified strain demonstrated a significantly higher mortality rate for the modified strain (80.0% vs. 52.0%, p = 0.037), with the odds of a lethal outcome being 3.7 times higher (OR = 3.69; 95% CI: 1.06–12.87) than for the wild-type strain.
Conclusions: Z. atratus larvae effectively fragment polystyrene but can serve as a reservoir for hyper-virulent pathogens. The induction of severe acute necrotizing pneumonia by plastic-adapted bacteria highlights the need for further biosafety assessments for by-products in insect-mediated recycling technologies.