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Role of RNAIII in Resistance to Antibiotics and Antimicrobial Agents in Staphylococcus epidermidis Biofilms.

ABSTRACT: Staphylococcus epidermidis is a known opportunistic pathogen and is one of the leading causes of chronic biofilm-associated infections. Biofilm formation is considered as a main strategy to resist antibiotic treatment and help bacteria escape from the human immune system. Understanding the complex mechanisms in biofilm formation can help find new ways to treat resistant strains and lower the prevalence of nosocomial infections. In order to examine the role of RNAIII regulated by the agr quorum sensing system and to what extent it influences biofilm resistance to antimicrobial agents, deletion mutant S. epidermidis RP62a-ΔRNAIII deficient in repressor domains with a re-maining functional hld gene was created. A deletion strain was used to examine the influence of oxacillin in combination with vanillin on biofilm resistance and cell survival was determined. Utilizing real-time qPCR, confocal laser scanning microscopy (CLSM), and crystal violet staining analyses, we found that the RNAIII-independent controlled phenol soluble modulins (PSMs) and RNAIII effector molecule have a significant role in biofilm resistance to antibiotics and phenolic compounds, and it protects the integrity of biofilms. Moreover, a combination of antibiotic and antimicrobial agents can induce methicillin-resistant S. epidermidis biofilm formation and can lead to exceedingly difficult medical treatment.

SUBMITTER: Minich A

PROVIDER: S-EPMC9569910 | biostudies-literature | 2022 Sep

REPOSITORIES: biostudies-literature

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Role of RNAIII in Resistance to Antibiotics and Antimicrobial Agents in Staphylococcus epidermidis Biofilms.

Minich Andrej A Lišková Veronika V Kormanová Ľubica Ľ Krahulec Ján J Šarkanová Júlia J Mikulášová Mária M Levarski Zdenko Z Stuchlík Stanislav S

International journal of molecular sciences 20220921 19

Staphylococcus epidermidis is a known opportunistic pathogen and is one of the leading causes of chronic biofilm-associated infections. Biofilm formation is considered as a main strategy to resist antibiotic treatment and help bacteria escape from the human immune system. Understanding the complex mechanisms in biofilm formation can help find new ways to treat resistant strains and lower the prevalence of nosocomial infections. In order to examine the role of RNAIII regulated by the ag ...[more]

PMID: 36232391

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Project description:Staphylococcus epidermidis is implicated in a multitude of human infections and is one of the major causes of clinical infections in hospitals, especially at surgical sites and on indwelling medical devices, such as orthopedic implants. These infections are especially dangerous because of the S. epidermidis propensity to form biofilms, which increases resistance to antibiotics and the natural immune response. This study investigated pulsed electromagnetic fields (PEMF) as a potential treatment to combat such infections, as PEMF exposure was expected to disrupt the electrostatic forces that adhere staphylococcal cells to surfaces and to one another. To test the effect of PEMF on biofilms, S. epidermidis cultures were exposed to PEMF at various durations either during the growth phase or after a full biofilm had formed. In addition, cells were exposed to PEMF and concomitant antibiotic treatment. Biofilm viability was quantified by both crystal violet and alamarBlue assays and scanning electron microscopy. The results demonstrated that PEMF significantly inhibited biofilm formation and disrupted preformed biofilms in vitro while also showing synergistic biofilm inhibition when combined with antibiotics. These combined results indicate that PEMF should be considered a promising novel technique for treating S. epidermidis biofilm infections and undergo further testing in vivo. IMPORTANCE Antibiotic resistance and biofilm infections are major issues in health care because of the lack of a successful treatment modality and poor patient outcomes. These infections are a particular issue following orthopedic surgery or trauma wherein an infection may form on an orthopedic implant or patient's bone. The presented study demonstrates that pulsed electromagnetic fields may be a promising novel treatment for such infections and can overcome the medical challenges presented by biofilm formation. Furthermore, the effects demonstrated are even greater when combining pulsed electromagnetic field therapy with traditional antibiotics.

Project description:Staphylococcus epidermidis is a leading opportunistic pathogen causing nosocomial infections that is notable for its ability to form a biofilm and for its high rates of antibiotic resistance. It serves as a reservoir of multiple antimicrobial resistance genes that spread among the staphylococcal population by horizontal gene transfer such as transduction. While phage-mediated transduction is well studied in Staphylococcus aureus, S. epidermidis transducing phages have not been described in detail yet. Here, we report the characteristics of four phages, 27, 48, 456, and 459, previously used for S. epidermidis phage typing, and the newly isolated phage E72, from a clinical S. epidermidis strain. The phages, classified in the family Siphoviridae and genus Phietavirus, exhibited an S. epidermidis-specific host range, and together they infected 49% of the 35 strains tested. A whole-genome comparison revealed evolutionary relatedness to transducing S. aureus phietaviruses. In accordance with this, all the tested phages were capable of transduction with high frequencies up to 10-4 among S. epidermidis strains from different clonal complexes. Plasmids with sizes from 4 to 19 kb encoding resistance to streptomycin, tetracycline, and chloramphenicol were transferred. We provide here the first evidence of a phage-inducible chromosomal island transfer in S. epidermidis Similarly to S. aureus pathogenicity islands, the transfer was accompanied by phage capsid remodeling; however, the interfering protein encoded by the island was distinct. Our findings underline the role of S. epidermidis temperate phages in the evolution of S. epidermidis strains by horizontal gene transfer, which can also be utilized for S. epidermidis genetic studies.IMPORTANCE Multidrug-resistant strains of S. epidermidis emerge in both nosocomial and livestock environments as the most important pathogens among coagulase-negative staphylococcal species. The study of transduction by phages is essential to understanding how virulence and antimicrobial resistance genes spread in originally commensal bacterial populations. In this work, we provide a detailed description of transducing S. epidermidis phages. The high transduction frequencies of antimicrobial resistance plasmids and the first evidence of chromosomal island transfer emphasize the decisive role of S. epidermidis phages in attaining a higher pathogenic potential of host strains. To date, such importance has been attributed only to S. aureus phages, not to those of coagulase-negative staphylococci. This study also proved that the described transducing bacteriophages represent valuable genetic modification tools in S. epidermidis strains where other methods for gene transfer fail.

Project description:In this paper, the metabolic activity in single and dual species biofilms of Staphylococcus epidermidis and Staphylococcus aureus isolates was investigated. Our results demonstrated that there was less metabolic activity in dual species biofilms compared to S. aureus biofilms. However, this was not observed if S. aureus and S. epidermidis were obtained from the same sample. The largest effect on metabolic activity was observed in biofilms of S. aureus Mu50 and S. epidermidis ET-024. A transcriptomic analysis of these dual species biofilms showed that urease genes and genes encoding proteins involved in metabolism were downregulated in comparison to monospecies biofilms. These results were subsequently confirmed by phenotypic assays. As metabolic activity is related to acid production, the pH in dual species biofilms was slightly higher compared to S. aureus Mu50 biofilms. Our results showed that S. epidermidis ET-024 in dual species biofilms inhibits metabolic activity of S. aureus Mu50, leading to less acid production. As a consequence, less urease activity is required to compensate for low pH. Importantly, this effect was biofilm-specific. Also S. aureus Mu50 genes encoding virulence-associated proteins (Spa, SplF and Dps) were upregulated in dual species biofilms compared to monospecies biofilms and using Caenorhabditis elegans infection assays, we demonstrated that more nematodes survived when co-infected with S. epidermidis ET-024 and S. aureus mutants lacking functional spa, splF or dps genes, compared to nematodes infected with S. epidermidis ET-024 and wild- type S. aureus. Finally, S. epidermidis ET-024 genes encoding resistance to oxacillin, erythromycin and tobramycin were upregulated in dual species biofilms and increased resistance was subsequently confirmed. Our data indicate that both species in dual species biofilms of S. epidermidis and S. aureus influence each other's behavior, but additional studies are required necessary to elucidate the exact mechanism(s) involved.

Project description:The ability of Staphylococcus epidermidis and S. aureus to form strong biofilm on plastic devices makes them the major pathogens associated with device-related infections (DRIs). Biofilm-embedded bacteria are more resistant to antibiotics, making biofilm infections very difficult to effectively treat. Here, we evaluate the in vitro activities of anti-staphylococcal drug oxacillin and antimicrobial peptide nisin, alone and in combination, against methicillin-resistant S. epidermidis (MRSE) clinical isolates and the methicillin-resistant S. aureus ATCC 43,300. The minimum inhibitory concentrations (MIC) and minimum biofilm eradication concentrations (MBEC) of oxacillin and nisin were determined using the microbroth dilution method. The anti-biofilm activities of oxacillin and nisin, alone or in combination, were evaluated. In addition, the effects of antimicrobial agents on the expression of icaA gene were examined by quantitative real-time PCR. MIC values for oxacillin and nisin ranged 4-8 µg/mL and 64-128 µg/mL, respectively. Oxacillin and nisin reduced biofilm biomass in all bacteria in a dose-dependent manner and this inhibitory effect was enhanced with combinatorial treatment. MBEC ranges for oxacillin and nisin were 2048-8192 µg/mL and 2048-4096 µg/mL, respectively. The addition of nisin significantly decreased the oxacillin MBECs from 8- to 32-fold in all bacteria. At the 1× MIC and 1/2× MIC, both oxacillin and nisin decreased significantly the expression of icaA gene in comparison with untreated control. When two antimicrobial agents were combined at 1/2× MIC concentration, the expression of icaA were significantly lower than when were used alone. Nisin/conventional oxacillin combination showed considerable anti-biofilm effects, including inhibition of biofilm formation, eradication of mature biofilm, and down-regulation of biofilm-related genes, proposing its applications for treating or preventing staphylococcal biofilm-associated infections, including device-related infections.

Project description:Biofilms on wound surfaces are treated topically with hyperosmotic agents, such as medical-grade honey and cadexomer iodine; in some cases, these treatments are combined with antibiotics. Tissue repair requires oxygen, and a low pH is conducive to oxygen release from red blood cells and epithelialization. We investigated the variation of dissolved oxygen concentration and pH with biofilm depth and the variation in oxygen consumption rates when biofilms are challenged with medical-grade honey or cadexomer iodine combined with vancomycin or ciprofloxacin. Dissolved oxygen and pH depth profiles in Staphylococcus aureus biofilms were measured using microelectrodes. The presence of cadexomer iodine with vancomycin or ciprofloxacin on the surface of the biofilm permitted a measurable concentration of oxygen at greater biofilm depths (101.6 ± 27.3 μm, P = 0.02; and 155.5 ± 27.9 μm, P = 0.016, respectively) than in untreated controls (30.1 μm). Decreases in pH of ∼0.6 and ∼0.4 units were observed in biofilms challenged with medical-grade honey alone and combined with ciprofloxacin, respectively (P < 0.001 and 0.01, respectively); the number of bacteria recovered from biofilms was significantly reduced (1.26 log) by treatment with cadexomer iodine and ciprofloxacin (P = 0.002) compared to the untreated control. Combining cadexomer iodine and ciprofloxacin improved dissolved oxygen concentration and penetration depth into the biofilm, while medical-grade honey was associated with a lower pH; not all treatments established a bactericidal effect in the time frame used in the experiments.IMPORTANCE Reports about using hyperosmotic agents and antibiotics against wound biofilms focus mostly on killing bacteria, but the results of these treatments should additionally be considered in the context of how they affect physiologically important parameters, such as oxygen concentration and pH. We confirmed that the combination of a hyperosmotic agent and an antibiotic results in greater dissolved oxygen and reduced pH within an S. aureus biofilm.

Project description:The microbiota influences host health through several mechanisms, including protecting it from pathogen colonization. Staphylococcus epidermidis is one of the most frequently found species in the skin microbiota, and its presence can limit the development of pathogens such as Staphylococcus aureusS. aureus causes diverse types of infections ranging from skin abscesses to bloodstream infections. Given the increasing prevalence of S. aureus drug-resistant strains, it is imperative to search for new strategies for treatment and prevention. Thus, we investigated the activity of molecules produced by a commensal S. epidermidis isolate against S. aureus biofilms. We showed that molecules present in S. epidermidis cell-free conditioned media (CFCM) caused a significant reduction in biofilm formation in most S. aureus clinical isolates, including all 4 agr types and agr-defective strains, without any impact on growth. S. epidermidis molecules also disrupted established S. aureus biofilms and reduced the antibiotic concentration required to eliminate them. Preliminary characterization of the active compound showed that its activity is resistant to heat, protease inhibitors, trypsin, proteinase K, and sodium periodate treatments, suggesting that it is not proteinaceous. RNA sequencing revealed that S. epidermidis-secreted molecules modulate the expression of hundreds of S. aureus genes, some of which are associated with biofilm production. Biofilm formation is one of the main virulence factors of S. aureus and has been associated with chronic infections and antimicrobial resistance. Therefore, molecules that can counteract this virulence factor may be promising alternatives as novel therapeutic agents to control S. aureus infections.IMPORTANCES. aureus is a leading agent of infections worldwide, and its main virulence characteristic is the ability to produce biofilms on surfaces such as medical devices. Biofilms are known to confer increased resistance to antimicrobials and to the host immune responses, requiring aggressive antibiotic treatment and removal of the infected surface. Here, we investigated a new source of antibiofilm compounds, the skin microbiome. Specifically, we found that a commensal strain of S. epidermidis produces molecules with antibiofilm activity, leading to a significant decrease of S. aureus biofilm formation and to a reduction of previously established biofilms. The molecules potentiated the activity of antibiotics and affected the expression of hundreds of S. aureus genes, including those associated with biofilm formation. Our research highlights the search for compounds that can aid us in the fight against S. aureus infections.

Dataset Information

Role of RNAIII in Resistance to Antibiotics and Antimicrobial Agents in Staphylococcus epidermidis Biofilms.

Publications

Role of RNAIII in Resistance to Antibiotics and Antimicrobial Agents in <i>Staphylococcus epidermidis</i> Biofilms.

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