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Staphylococcus aureus interaction with Pseudomonas aeruginosa biofilm enhances tobramycin resistance.

ABSTRACT: Antimicrobial resistance is a significant threat to the treatment of infectious disease. Multiple mechanisms of resistance to different classes of antibiotics have been identified and well-studied. However, these mechanisms are studied with bacteria in isolation, whereas often, infections have a polymicrobial basis. Using a biofilm slide chamber model, we visualized the formation and development of clinical Pseudomonas aeruginosa biofilms in the presence of secreted Staphylococcus aureus exoproducts, two bacteria that commonly co-infect pediatric patients with cystic fibrosis. We showed that, over time, certain isolates of P. aeruginosa can form different biofilm architecture in the presence of S. aureus exoproducts. We further determined that this interaction was dependent on Psl produced by P. aeruginosa and staphylococcal protein A from S. aureus. Importantly, we identified a mechanism of antibiotic resistance to tobramycin that is dependent on the polymicrobial interactions between these two bacteria. This interaction occurred in isolates of P. aeruginosa recovered from children with cystic fibrosis who failed to clear P. aeruginosa following inhaled tobramycin treatment.

SUBMITTER: Beaudoin T

PROVIDER: S-EPMC5648753 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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Staphylococcus aureus interaction with Pseudomonas aeruginosa biofilm enhances tobramycin resistance.

Beaudoin T T Yau Y C W YCW Stapleton P J PJ Gong Y Y Wang P W PW Guttman D S DS Waters V V

NPJ biofilms and microbiomes 20171019

Antimicrobial resistance is a significant threat to the treatment of infectious disease. Multiple mechanisms of resistance to different classes of antibiotics have been identified and well-studied. However, these mechanisms are studied with bacteria in isolation, whereas often, infections have a polymicrobial basis. Using a biofilm slide chamber model, we visualized the formation and development of clinical Pseudomonas aeruginosa biofilms in the presence of secreted Staphylococcus aure ...[more]

PMID: 29062489

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Project description:Cystic fibrosis (CF) is a human genetic disorder which results in a lung environment that is highly conducive to chronic microbial infection. Over the past decade, deep-sequencing studies have demonstrated that the CF lung can harbor a highly diverse polymicrobial community. We expanded our existing in vitro model of Pseudomonas aeruginosa biofilm formation on CF-derived airway cells to include this broader set of CF airway colonizers to investigate their contributions to CF lung disease, particularly as they relate to the antibiotic response of the population. Using this system, we identified an interspecies interaction between P. aeruginosa, a bacterium associated with declining lung function and worsening disease, and Streptococcus constellatus, a bacterium correlated with the onset of pulmonary exacerbations in CF patients. The growth rate and cytotoxicity of S. constellatus 7155 and P. aeruginosa PA14 were unchanged when grown together as mixed biofilms in the absence of antibiotics. However, the addition of tobramycin, the frontline maintenance therapy antibiotic for individuals with CF, to a mixed biofilm of S. constellatus 7155 and P. aeruginosa PA14 resulted in enhanced S. constellatus biofilm formation. Through a candidate genetic approach, we showed that P. aeruginosa rhamnolipids were reduced upon tobramycin exposure, allowing for S. constellatus 7155 biofilm enhancement, and monorhamnolipids were sufficient to reduce S. constellatus 7155 biofilm viability in the absence of tobramycin. While the findings presented here are specific to a biofilm of S. constellatus 7155 and P. aeruginosa PA14, they highlight the potential of polymicrobial interactions to impact antibiotic tolerance in unanticipated ways.Deep-sequencing studies have demonstrated that the CF lung can harbor a diverse polymicrobial community. By recapitulating the polymicrobial communities observed in the CF lung and identifying mechanisms of interspecies interactions, we have the potential to select the best therapy for a given bacterial community and reveal potential opportunities for novel therapeutic interventions. Using an in vitro model of bacterial infection on CF airway cells, we tested how a particular polymicrobial community grows, damages human cells, and responds to antibiotics in single and mixed infections. We describe here the mechanism of an interspecies interaction between two pathogens in the CF lung, P. aeruginosa and S. constellatus, which is potentiated by a commonly prescribed antibiotic, tobramycin.

Project description:The Pseudomonas aeruginosa LasR-LasI (LasR-I) quorum sensing system regulates secreted proteases that can be exploited by cheaters, such as quorum sensing receptor-defective (lasR) mutants. lasR mutants emerge in populations growing on casein as a sole source of carbon and energy. These mutants are exploitative cheaters because they avoid the substantial cost of engaging in quorum sensing. Previous studies showed that quorum sensing increases resistance to some antibiotics, such as tobramycin. Here, we show that tobramycin suppressed the emergence of lasR mutants in casein-passaged populations. Several mutations accumulated in those populations, indicating evidence of antibiotic adaptation. We found that mutations in one gene, ptsP, increased antibiotic resistance and also pleiotropically increased production of a quorum sensing-controlled phenazine, pyocyanin. When passaged on casein, ptsP mutants suppressed cheaters in a manner that was tobramycin independent. We found that the mechanism of cheater suppression in ptsP mutants relied on pyocyanin, which acts as a policing toxin by selectively blocking growth of cheaters. Thus, tobramycin suppresses lasR mutants through two mechanisms: first, through direct effects on cheaters and, second, by selecting mutations in ptsP that suppressed cheating in a tobramycin-independent manner. This work demonstrates how adaptive mutations can alter the dynamics of cooperator-cheater relationships, which might be important for populations adapting to antibiotics during interspecies competition or infections. IMPORTANCE The opportunistic pathogen Pseudomonas aeruginosa is a model for understanding quorum sensing, a type of cell-cell signaling important for cooperation. Quorum sensing controls production of cooperative goods, such as exoenzymes, which are vulnerable to cheating by quorum sensing-defective mutants. Because uncontrolled cheating can ultimately cause a population to collapse, much focus has been on understanding how P. aeruginosa can control cheaters. We show that an antibiotic, tobramycin, can suppress cheaters in cooperating P. aeruginosa populations. Tobramycin suppresses cheaters directly because the cheaters are more susceptible to tobramycin than cooperators. Tobramycin also selects for mutations in a gene, ptsP, that suppresses cheaters independent of tobramycin through pleiotropic regulation of a policing toxin, pyocyanin. This work supports the idea that adaptation to antibiotics can have unexpected effects on the evolution of quorum sensing and has implications for understanding how cooperation evolves in dynamic bacterial communities.

Project description:ObjectivesStaphylococcus aureus and Pseudomonas aeruginosa were the most common bacteria in nosocomial infections. Different bacteriocins are currently being studied as antibiotics or in conjunction with antibiotics as potential strategies to treat resistant infectious agents. The study aimed to determine nisin's effect on the biofilm production, antimicrobial susceptibility, and biofilm formation of S. aureus and P. aeruginosa.Materials and methodsThe experimental research tested two antibiotic-resistant isolates of S. aureus and P. aeruginosa strains. The experimental study tested two antibiotic-resistant isolates of S. aureus and P. aeruginosa strains. The MIC of bacteriocin nisin was determined using the micro broth dilution method, and crystal violet was used to assess the effect of bacteriocin on the biofilm. In addition, L929 cell culture was used to determine the effectiveness of bacteriocin on the isolate under similar cell conditions. Moreover, the MTT assay was used to and evaluate bacteriocin toxicity. In this study, the software Prism version 9 and Graph pad software were utilized.ResultsThe results of this study reveal that the nisin has different activities at different doses and is considered dose-dependent. At various times and doses, nisin inhibits biofilm formation in S. aureus, and P. aeruginosa isolates. Nisin also showed a decreasing survival of the isolates. Antibiotic-resistant bacteria can be made more vulnerable by nisin. Furthermore, nisin treatment affected the production of virulence factors such as hemolysins in S. aureus and had little or a negative effect on P. aeruginosa virulence factors. This medication stops S. aureus and P. aeruginosa from growing and causes bacterial cell damage.ConclusionsAntibacterial properties of nicin against S. aureus and P. aeruginosa were successfully studied. This bacteriocin stops S. aureus and P. aeruginosa from growing and causes bacterial cell damage or death. Damage to the membrane among the fundamental causes is reduced membrane potential and enzyme inactivation.

Project description:The airways of cystic fibrosis (CF) patients have thick mucus, which fosters chronic, polymicrobial infections. Pseudomonas aeruginosa and Staphylococcus aureus are two of the most prevalent respiratory pathogens in CF patients. In this study, we tested whether P. aeruginosa influences the susceptibility of S. aureus to frontline antibiotics used to treat CF lung infections. Using our in vitro coculture model, we observed that addition of P. aeruginosa supernatants to S. aureus biofilms grown either on epithelial cells or on plastic significantly decreased the susceptibility of S. aureus to vancomycin. Mutant analyses showed that 2-n-heptyl-4-hydroxyquinoline N-oxide (HQNO), a component of the P. aeruginosa Pseudomonas quinolone signal (PQS) system, protects S. aureus from the antimicrobial activity of vancomycin. Similarly, the siderophores pyoverdine and pyochelin also contribute to the ability of P. aeruginosa to protect S. aureus from vancomycin, as did growth under anoxia. Under our experimental conditions, HQNO, P. aeruginosa supernatant, and growth under anoxia decreased S. aureus growth, likely explaining why this cell wall-targeting antibiotic is less effective. P. aeruginosa supernatant did not confer additional protection to slow-growing S. aureus small colony variants. Importantly, P. aeruginosa supernatant protects S. aureus from other inhibitors of cell wall synthesis as well as protein synthesis-targeting antibiotics in an HQNO- and siderophore-dependent manner. We propose a model whereby P. aeruginosa causes S. aureus to shift to fermentative growth when these organisms are grown in coculture, leading to reduction in S. aureus growth and decreased susceptibility to antibiotics targeting cell wall and protein synthesis.IMPORTANCE Cystic fibrosis (CF) lung infections are chronic and difficult to eradicate. Pseudomonas aeruginosa and Staphylococcus aureus are two of the most prevalent respiratory pathogens in CF patients and are associated with poor patient outcomes. Both organisms adopt a biofilm mode of growth, which contributes to high tolerance to antibiotic treatment and the recalcitrant nature of these infections. Here, we show that P. aeruginosa exoproducts decrease the sensitivity of S. aureus biofilm and planktonic populations to vancomycin, a frontline antibiotic used to treat methicillin-resistant S. aureus in CF patients. P. aeruginosa also protects S. aureus from other cell wall-active antibiotics as well as various classes of protein synthesis inhibitors. Thus, interspecies interactions can have dramatic and unexpected consequences on antibiotic sensitivity. This study underscores the potential impact of interspecies interactions on antibiotic efficacy in the context of complex, polymicrobial infections.

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Staphylococcus aureus interaction with Pseudomonas aeruginosa biofilm enhances tobramycin resistance.

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<i>Staphylococcus aureus</i> interaction with <i>Pseudomonas aeruginosa</i> biofilm enhances tobramycin resistance.

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