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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:Pseudomonas aeruginosa and Staphylococcus aureus often cause chronic, recalcitrant infections in large part due to their ability to form biofilms. The biofilm mode of growth enables these organisms to withstand antibacterial insults that would effectively eliminate their planktonic counterparts. We found that P. aeruginosa supernatant increased the sensitivity of S. aureus biofilms to multiple antimicrobial compounds, including fluoroquinolones and membrane-targeting antibacterial agents, including the antiseptic chloroxylenol. Treatment of S. aureus with the antiseptic chloroxylenol alone did not decrease biofilm cell viability; however, the combination of chloroxylenol and P. aeruginosa supernatant led to a 4-log reduction in S. aureus biofilm viability compared to exposure to chloroxylenol alone. We found that the P. aeruginosa-produced small molecule 2-n-heptyl-4-hydroxyquinoline N-oxide (HQNO) is responsible for the observed heightened sensitivity of S. aureus to chloroxylenol. Similarly, HQNO increased the susceptibility of S. aureus biofilms to other compounds, including both traditional and nontraditional antibiotics, which permeabilize bacterial membranes. Genetic and phenotypic studies support a model whereby HQNO causes an increase in S. aureus membrane fluidity, thereby improving the efficacy of membrane-targeting antiseptics and antibiotics. Importantly, our data show that P. aeruginosa exoproducts can enhance the ability of various antimicrobial agents to kill biofilm populations of S. aureus that are typically difficult to eradicate. Finally, our discovery that altering membrane fluidity shifts antimicrobial sensitivity profiles of bacterial biofilms may guide new approaches to target persistent infections, such as those commonly found in respiratory tract infections and in chronic wounds.IMPORTANCE The thick mucus in the airways of cystic fibrosis (CF) patients predisposes them to frequent, polymicrobial respiratory infections. Pseudomonas aeruginosa and Staphylococcus aureus are frequently coisolated from the airways of individuals with CF, as well as from diabetic foot ulcers and other wounds. Both organisms form biofilms, which are notoriously difficult to eradicate and promote chronic infection. In this study, we have shown that P. aeruginosa-secreted factors can increase the efficacy of compounds that alone have little or no bactericidal activity against S. aureus biofilms. In particular, we discovered that P. aeruginosa exoproducts can potentiate the antistaphylococcal activity of phenol-based antiseptics and other membrane-active drugs. Our findings illustrate that polymicrobial interactions can dramatically increase antibacterial efficacy in vitro and suggest that altering membrane physiology promotes the ability of certain drugs to kill bacterial biofilms-knowledge that may provide a path for the discovery of new biofilm-targeting antimicrobial strategies.

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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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