# Coexistence with Staphylococcus aureus modulates the virulence and antibiotic resistance of Pseudomonas aeruginosa

## TL;DR

Coexistence with Staphylococcus aureus increases Pseudomonas aeruginosa's antibiotic resistance and virulence in both lab and mouse experiments.

## Contribution

This study reveals that co-culturing P. aeruginosa with S. aureus enhances resistance and virulence through synergistic interactions.

## Key findings

- Co-cultured P. aeruginosa showed increased survival when treated with multiple antibiotics.
- Virulence factors like pyocyanin and biofilm production were significantly elevated in co-cultures.
- In vivo experiments showed co-infection with S. aureus increased P. aeruginosa pathogenicity and mortality in mice.

## Abstract

In this study, we were aimed to investigate the impact of co-cultures of different bacterial species on bacterial antibiotic resistance and virulence.

The effect of co-cultures of Pseudomonas aeruginosa (Gram-negative bacteria) and Staphylococcus aureus (Gram-positive bacteria) on antibiotic resistance, virulence and biofilm formation in P. aeruginosa was examined in vitro in 14 mixtures. These mixtures were categorized into three groups: Standard category (including standard strains), naturally co-isolated category (co-isolated from the same patient) and random co-culture category (bacterial species from different patients). Additionally, the impact of the standard category on pathogenicity was assessed in vivo using mouse model. Intergroup comparisons were conducted using multiple t-test and comparisons between treated and untreated control isolates grown under the same conditions were made. Survival experiments were analyzed using Mantel-Cox log-rank test.

P. aeruginosa survival significantly increased in most of the co-culture mixtures when treated with meropenem (92.9%), ceftazidime (85.7%), cefepime (78.6%), gentamicin (78.6%) and ciprofloxacin (71.4%). Similarly, virulence factor production significantly increased in P. aeruginosa in most of the investigated co-cultures as follows: pyocyanin (71.4%), elastase (71.4%), protease (78.6%), hemolysin (71.4%), lecithinase (78.6%), gelatinase (63.6%) and biofilm (71.4%). At the molecular level, the relative expression of the tested virulence-encoding genes; pelA, lasB and lasA were significantly increased in at least 92.9% of the co-culture mixtures, especially in random ones, compared to their mono-culture, but with varying up-regulation degree (ranging from 1.5 to 96-fold increase).

Finally, in vitro investigations for antibiotic resistance and virulence production clearly demonstrated a synergistic interaction between P. aeruginosa and S. aureus in the co-existence mixture. Compared to P. aeruginosa mono-cultures, the co-cultured strains exhibited enhanced resistance profiles and increased expression of key virulence factors, indicating that the simultaneous presence of both species promotes mutual adaptation and potentiation of pathogenic traits. Additionally, in vivo experiments confirmed that the co-infection with S. aureus significantly enhanced the pathogenicity of P. aeruginosa, as indicated by increased mortality rates and higher bacterial counts in lung tissues. Altogether, our results shed light on the impact of the co-existence of microbial species on bacterial virulence and antibiotic resistance.

The online version contains supplementary material available at 10.1186/s12941-025-00843-2.