Type III-A CRISPR immunity promotes mutagenesis of staphylococci.

Horizontal gene transfer and mutation are the two major drivers of microbial evolution that enable bacteria to adapt to fluctuating environmental stressors1. Clustered, regularly interspaced, short palindromic repeats (CRISPR) systems use RNA-guided nucleases to direct sequence-specific destruction of the genomes of mobile genetic elements that mediate horizontal gene transfer, such as conjugative plasmids2 and bacteriophages3, thus limiting the extent to which bacteria can evolve by this mechanism. A subset of CRISPR systems also exhibit non-specific degradation of DNA4,5; however, whether and how this feature affects the host has not yet been examined. Here we show that the non-specific DNase activity of the staphylococcal type III-A CRISPR-Cas system increases mutations in the host and accelerates the generation of antibiotic resistance in Staphylococcus aureus and Staphylococcus epidermidis. These mutations require the induction of the SOS response to DNA damage and display a distinct pattern. Our results demonstrate that by differentially affecting both mechanisms that generate genetic diversity, type III-A CRISPR systems can modulate the evolution of the bacterial host.

Biosafety of human environments can be supported by effective use of renewable biomass.

Preventing pathogenic viral and bacterial transmission in the human environment is critical, especially in potential outbreaks that may be caused by the release of ancient bacteria currently trapped in the permafrost. Existing commercial disinfectants present issues such as a high carbon footprint. This study proposes a sustainable alternative, a bioliquid derived from biomass prepared by hydrothermal liquefaction. Results indicate a high inactivation rate of pathogenic virus and bacteria by the as-prepared bioliquid, such as up to 99.99% for H1N1, H5N1, H7N9 influenza A virus, and Bacillus subtilis var. niger spores and 99.49% for Bacillus anthracis Inactivation of Escherichia coli and Staphylococcus epidermidis confirmed that low-molecular-weight and low-polarity compounds in bioliquid are potential antibacterial components. High temperatures promoted the production of antibacterial substances via depolymerization and dehydration reactions. Moreover, bioliquid was innoxious as confirmed by the rabbit skin test, and the cost per kilogram of the bioliquid was $0.04427, which is notably lower than that of commercial disinfectants. This study demonstrates the potential of biomass to support our biosafety with greater environmental sustainability.

Immune checkpoint inhibitors unleash pathogenic immune responses against the microbiota.

Immune checkpoint inhibitors (ICIs) are essential components of the cancer therapeutic armamentarium. While ICIs have demonstrated remarkable clinical responses, they can be accompanied by immune-related adverse events (irAEs). These inflammatory side effects are of unclear etiology and impact virtually all organ systems, with the most common being sites colonized by the microbiota such as the skin and gastrointestinal tract. Here, we establish a mouse model of commensal bacteria-driven skin irAEs and demonstrate that immune checkpoint inhibition unleashes commensal-specific inflammatory T cell responses. These aberrant responses were dependent on production of IL-17 by commensal-specific T cells and induced pathology that recapitulated the cutaneous inflammation seen in patients treated with ICIs. Importantly, aberrant T cell responses unleashed by ICIs were sufficient to perpetuate inflammatory memory responses to the microbiota months following the cessation of treatment. Altogether, we have established a mouse model of skin irAEs and reveal that ICIs unleash aberrant immune responses against skin commensals, with long-lasting inflammatory consequences.

Photobiocidal Activity of TiO2/UHMWPE Composite Activated by Reduced Graphene Oxide under White Light.

Herein, we introduce a photobiocidal surface activated by white light. The photobiocidal surface was produced through thermocompressing a mixture of titanium dioxide (TiO2), ultra-high-molecular-weight polyethylene (UHMWPE), and reduced graphene oxide (rGO) powders. A photobiocidal activity was not observed on UHMWPE-TiO2. However, UHMWPE-TiO2@rGO exhibited potent photobiocidal activity (>3-log reduction) against Staphylococcus epidermidis and Escherichia coli bacteria after a 12 h exposure to white light. The activity was even more potent against the phage phi 6 virus, a SARS-CoV-2 surrogate, with a >5-log reduction after 6 h exposure to white light. Our mechanistic studies showed that the UHMWPE-TiO2@rGO was activated only by UV light, which accounts for 0.31% of the light emitted by the white LED lamp, producing reactive oxygen species that are lethal to microbes. This indicates that adding rGO to UHMWPE-TiO2 triggered intense photobiocidal activity even at shallow UV flux levels.

Synthetic Peptides Capable of Potent Multigroup Staphylococcal Quorum Sensing Activation and Inhibition in Both Cultures and Biofilm Communities.

The pathogen Staphylococcus epidermidis uses a chemical signaling process, i.e., quorum sensing (QS), to form robust biofilms and cause human infection. Many questions remain about QS in S. epidermidis, as it uses this intercellular communication pathway to both negatively and positively regulate virulence traits. Herein, we report synthetic multigroup agonists and antagonists of the S. epidermidis accessory gene regulator (agr) QS system capable of potent superactivation and complete inhibition, respectively. These macrocyclic peptides maintain full efficacy across the three major agr specificity groups, and their activity can be "mode-switched" from agonist to antagonist via subtle residue-specific structural changes. We describe the design and synthesis of these non-native peptides and demonstrate that they can appreciably decrease biofilm formation on abiotic surfaces, underscoring the potential for agr agonism as a route to block S. epidermidis virulence. Additionally, we show that both the S. epidermidis agonists and antagonists are active in S. aureus, another common pathogen with a related agr system, yet only as antagonists. This result not only revealed one of the most potent agr inhibitors known in S. aureus but also highlighted differences in the mechanisms of agr agonism and antagonism between these related bacteria. Finally, our investigations reveal unexpected inhibitory behavior for certain S. epidermidis agr agonists at sub-activating concentrations, an observation that can be leveraged for the design of future probes with enhanced potencies. Together, these peptides provide a powerful tool set to interrogate the role of QS in S. epidermidis infections and in Staphylococcal pathogenicity in general.

Dual Antibacterial Properties of Copper-Coated Nanotextured Stainless Steel.

Bacterial adhesion to stainless steel, an alloy commonly used in shared settings, numerous medical devices, and food and beverage sectors, can give rise to serious infections, ultimately leading to morbidity, mortality, and significant healthcare expenses. In this study, Cu-coated nanotextured stainless steel (nSS) fabrication have been demonstrated using electrochemical technique and its potential as an antibiotic-free biocidal surface against Gram-positive and negative bacteria. As nanotexture and Cu combine for dual methods of killing, this material should not contribute to drug-resistant bacteria as antibiotic use does. This approach involves applying a Cu coating on nanotextured stainless steel, resulting in an antibacterial activity within 30 min. Comprehensive characterization of the surface revealing that the Cu coating consists of metallic Cu and oxidized states (Cu2+ and Cu+), has been performed by this study. Cu-coated nSS induces a remarkable reduction of 97% in Gram-negative Escherichia coli and 99% Gram-positive Staphylococcus epidermidis bacteria. This material has potential to be used to create effective, scalable, and sustainable solutions to prevent bacterial infections caused by surface contamination without contributing to antibiotic resistance.

Comparison of the in vitro activities of delafloxacin and comparators against Staphylococcus epidermidis clinical strains involved in osteoarticular infections: a CRIOGO multicentre retrospective study.

OBJECTIVES: Staphylococcus epidermidis bone and joint infections (BJIs) on material are often difficult to treat. The activity of delafloxacin has not yet been studied on S. epidermidis in this context. The aim of this study was to assess its in vitro activity compared with other fluoroquinolones, against a large collection of S. epidermidis clinical strains. METHODS: We selected 538 S. epidermidis strains isolated between January 2015 and February 2023 from six French teaching hospitals. One hundred and fifty-two strains were ofloxacin susceptible and 386 were ofloxacin resistant. Identifications were performed by MS and MICs were determined using gradient concentration strips for ofloxacin, levofloxacin, moxifloxacin and delafloxacin. RESULTS: Ofloxacin-susceptible strains were susceptible to all fluoroquinolones. Resistant strains had higher MICs of all fluoroquinolones. Strains resistant to ofloxacin (89.1%) still showed susceptibility to delafloxacin when using the Staphylococcus aureus 2021 CA-SFM/EUCAST threshold of 0.25 mg/L. In contrast, only 3.9% of the ofloxacin-resistant strains remained susceptible to delafloxacin with the 0.016 mg/L S. aureus breakpoint according to CA-SFM/EUCAST guidelines in 2022. The MIC50 was 0.094 mg/L and the MIC90 was 0.38 mg/L. CONCLUSIONS: We showed low delafloxacin MICs for ofloxacin-susceptible S. epidermidis strains and a double population for ofloxacin-resistant strains. Despite the absence of breakpoints for S. epidermidis, delafloxacin may be an option for the treatment of complex BJI, including strains with MICs of ≤0.094 mg/L, leading to 64% susceptibility. This study underlines the importance for determining specific S. epidermidis delafloxacin breakpoints for the management of BJI on material.

Probing fosfomycin's potential: a study on susceptibility testing and resistance in Staphylococcus epidermidis from prosthetic joint infections.

BACKGROUND: There are limited treatment options for prosthetic joint infections (PJI) due to multidrug-resistant Staphylococcus epidermidis (MDRSE). Fosfomycin (FOF) has gained attention as a potential therapy, but there is a paucity of information on the phenotypic and genotypic susceptibility amongst S. epidermidis, including MDRSE. OBJECTIVES: To investigate phenotypical and genotypical susceptibility to FOF in S. epidermidis isolates prospectively collected from PJIs in Sweden. METHODS: MIC determination was performed using in-house agar dilution (AD) and a commercial AD panel. Genes and gene variants associated with FOF resistance were analysed. RESULTS: Multidrug resistance was common [74/89 (83%) isolates were MDRSE].FOF inhibited all isolates except one, which had an MIC > 256 mg/L. The commercial AD panel demonstrated good overall performance but tended to overestimate the MIC, resulting in 84% essential agreement with the gold standard. Genomic analysis with publically available tools for whole-genome sequencing (WGS) data suggested genotypic FOF resistance in all isolates, but in-depth analysis revealed that fosB, associated with FOF resistance, was only present in the phenotypically resistant isolate. No other genes or gene variants associated with FOF resistance were detected. CONCLUSIONS: Phenotypic resistance to FOF and presence of fosB were rare in this collection, indicating FOF's potential as a treatment option for S. epidermidis. The commercial AD panel demonstrated high reproducibility, but EA with the reference method was less than optimal. Findings of genotypic FOF resistance using common tools for WGS data should be critically evaluated and appropriately verified with relevant fosB references for S. epidermidis.

The two-component regulatory systems GraRS and SrrAB mediate Staphylococcus aureus susceptibility to Pep5 produced by clinical isolate of Staphylococcus epidermidis.

Staphylococcus aureus is a common bacterium on the skin and in the nose that sometimes causes severe illness. Bacteriocins, antimicrobial peptides, or proteins produced by bacteria are candidates for the treatment of S. aureus infection. In this study, we found that a clinical Staphylococcus epidermidis strain, KSE112, produced the lantibiotic Pep5, which showed anti-S. aureus activity. The complete nucleotide sequence of the Pep5-encoding plasmid was determined. Several S. aureus two-component regulatory systems (TCSs) are known to be involved in bacteriocin susceptibility. Therefore, susceptibility tests were performed using TCS-inactivated S. aureus mutants to determine which TCS is responsible for Pep5 susceptibility; the ΔgraRS mutant exhibited increased susceptibility to Pep5, while the ΔsrrAB mutant exhibited decreased susceptibility. GraRS is known to regulate dltABCD and mprF in concert with vraFG, and Pep5 susceptibility was significantly increased in the ΔdltABCD, ΔmprF, and ΔvraFG mutants. Regarding the ΔsrrAB mutant, cross-resistance to aminoglycosides was observed. As aminoglycoside activity is known to be affected by aerobic respiration, we focused on qoxABCD and cydAB, which are quinol oxidase genes that are necessary for aerobic respiration and have downregulated the expression in the ΔsrrAB mutant. We constructed ΔqoxABCD and ΔcydAB mutants and found that qoxABCD inactivation decreased susceptibility to Pep5 and aminoglycosides. These results indicate that reduced aerobic respiration due to the reduced qoxABCD expression in the ΔsrrAB mutant decreased Pep5 activity.IMPORTANCEThe emergence of drug-resistant bacteria, including MRSA, is a severe health problem worldwide. Thus, the development of novel antimicrobial agents, including bacteriocins, is needed. In this report, we found a Pep5-producing strain with anti-S. aureus activity. We determined the complete sequence of the Pep5-encoding plasmid for the first time. However, in S. aureus, GraRS and its effectors conferred decreased susceptibility to Pep5. We also revealed that another TCS, SrrAB, affects susceptibility Pep5 and other lantibiotics by controlling aerobic respiration. In our study, we investigated the efficacy of Pep5 against S. aureus and other Gram-positive bacteria and revealed that respiratory constancy regulated by TCS is required for the antimicrobial activity of nisin, nukacin, and Pep5. These findings provide important information for the clinical application of bacteriocins and suggest that they have different properties among similar pore-forming lantibiotics.

Completed genome and emergence scenario of the multidrug-resistant nosocomial pathogen Staphylococcus epidermidis ST215.

BACKGROUND: A multidrug-resistant lineage of Staphylococcus epidermidis named ST215 is a common cause of prosthetic joint infections and other deep surgical site infections in Northern Europe, but is not present elsewhere. The increasing resistance among S. epidermidis strains is a global concern. We used whole-genome sequencing to characterize ST215 from healthcare settings. RESULTS: We completed the genome of a ST215 isolate from a Swedish hospital using short and long reads, resulting in a circular 2,676,787 bp chromosome and a 2,326 bp plasmid. The new ST215 genome was placed in phylogenetic context using 1,361 finished public S. epidermidis reference genomes. We generated 10 additional short-read ST215 genomes and 11 short-read genomes of ST2, which is another common multidrug-resistant lineage at the same hospital. We studied recombination's role in the evolution of ST2 and ST215, and found multiple recombination events averaging 30-50 kb. By comparing the results of antimicrobial susceptibility testing for 31 antimicrobial drugs with the genome content encoding antimicrobial resistance in the ST215 and ST2 isolates, we found highly similar resistance traits between the isolates, with 22 resistance genes being shared between all the ST215 and ST2 genomes. The ST215 genome contained 29 genes that were historically identified as virulence genes of S. epidermidis ST2. We established that in the nucleotide sequence stretches identified as recombination events, virulence genes were overrepresented in ST215, while antibiotic resistance genes were overrepresented in ST2. CONCLUSIONS: This study features the extensive antibiotic resistance and virulence gene content in ST215 genomes. ST215 and ST2 lineages have similarly evolved, acquiring resistance and virulence through genomic recombination. The results highlight the threat of new multidrug-resistant S. epidermidis lineages emerging in healthcare settings.

Antibiofilm activity of truncated Staphylococcus aureus phenol soluble modulin α2 (SaΔ1Δ2PSMα2) against Candida auris in vitro and in an animal model of catheter-associated infection.

Candida auris has emerged as a major multidrug-resistant nosocomial pathogen. The organism exhibits a persistent colonising phenotype, and causes recalcitrant infections often strongly linked to biofilm formation. Alternate strategies are urgently needed to combat this yeast and its biofilm-associated phenotype. This work aimed to evaluate the efficacy of select staphylococcal phenol soluble modulins (PSMs), namely, a truncated version of Staphylococcus aureus PSMα2 shortened by two amino acids at the N-terminal (SaΔ1Δ2PSMα2) and Staphylococcus epidermidis PSMδ against C. auris in vitro and in vivo. The antifungal and antibiofilm activity was tested by broth microdilution and XTT dye reduction assay. Combination effect with antifungal drugs was determined by fractional inhibitory concentration test. The efficacy of combination therapy using SaΔ1Δ2PSMα2 with amphotericin B or caspofungin was evaluated in murine model of C. auris catheter-associated infection. Based on antifungal activity, antibiofilm activity and cytotoxicity data, SaΔ1Δ2PSMα2 exhibited promising activity against C. auris biofilms. Nearly 50 % inhibition in biofilm formation was noted with 0.5-2 µM of the peptide against multiple clinical and C. auris colonizing isolates. It was synergistic with amphotericin B (ΣFIC = 0.281) and caspofungin (ΣFIC = 0.047) in vitro, and improved the activity of voriconazole in voriconazole-resistant C. auris. Combination therapy using amphotericin B or caspofungin (1 µg/ml) with SaΔ1Δ2PSMα2 resulted in 99.5 % reduction in C. auris biofilm in murine model, even when the peptide was used at a concentration that was neither fungicidal nor antibiofilm (0.125 µM; ≈0.26 µg/ml). The study provides insight into the potential utility of SaΔ1Δ2PSMα2-antifungal drug combination against C. auris biofilm-associated infections.

Control of pathogenic bacterial biofilm associated with acne and the anti-inflammatory potential of an essential oil blend.

Acne is one of the most common skin conditions worldwide, with multifactorial origins it affects areas of the skin with hair follicles and sebaceous glands that become clogged. Bacterial incidence aggravates treatment due to resistance to antimicrobial agents and production of virulence factors such as biofilm formation. Based on these information, this study aims to conduct in vitro evaluations of the antibacterial activity of essential oils (EOs), alone and in combination, against Propionibacterium acnes, Staphylococcus aureus, and Staphylococcus epidermidis in planktonic and biofilm forms. This study also assessed the anti-inflammatory potential (TNF-α) and the effects of EOs on the viability of human keratinocytes (HaCaT), murine fibroblasts (3T3-L1), and bone marrow-derived macrophages (BMDMs). Of all EOs tested, 13 had active action against P. acnes, 9 against S. aureus, and 9 against S. epidermidis at concentrations of 0.125-2.0 mg/mL. Among the most active plant species, a blend of essential oil (BEOs) was selected, with Cymbopogon martini (Roxb.) Will. Watson, Eugenia uniflora L., and Varronia curassavica Jacq., the latter due to its anti-inflammatory action. This BEOs showed higher inhibition rates when compared to chloramphenicol against S. aureus and S. epidermidis, and higher eradication rates when compared to chloramphenicol for the three target species. The BEOs did not affect the cell viability of cell lines evaluated, and the levels of TNF-α decreased. According to these results, the BEOs evaluated showed potential for the development of an alternative natural formulation for the treatment of acne.

Bioemulsifier from sponge-associated bacteria reduces staphylococcal biofilm.

Biofilm formation is a major health concern and studies have been pursued to find compounds able to prevent biofilm establishment and remove pre-existing biofilms. While biosurfactants (BS) have been well-known for possessing antibiofilm activities, bioemulsifiers (BE) are still scarcely explored for this purpose. The present study aimed to evaluate the bioemulsifying properties of cell-free supernatants produced by Bacillaceae and Vibrio strains isolated from marine sponges and investigate their antiadhesive and antibiofilm activities against different pathogenic Gram-positive and Gram-negative bacteria. The BE production by the marine strains was confirmed by the emulsion test, drop-collapsing, oil-displacement, cell hydrophobicity and hemolysis assays. Notably, Bacillus cereus 64BHI1101 displayed remarkable emulsifying activity and the ultrastructure analysis of its BE extract (BE64-1) revealed the presence of structures typically observed in macromolecules composed of polysaccharides and proteins. BE64-1 showed notable antiadhesive and antibiofilm activities against Staphylococcus aureus, with a reduction of adherence of up to 100 % and a dispersion of biofilm of 80 %, without affecting its growth. BE64-1 also showed inhibition of Staphylococcus epidermidis and Escherichia coli biofilm formation and adhesion. Thus, this study provides a starting point for exploring the antiadhesive and antibiofilm activities of BE from sponge-associated bacteria, which could serve as a valuable tool for future research to combat S. aureus biofilms.

Diclofenac sodium effectively inhibits the biofilm formation of Staphylococcus epidermidis.

Staphylococcus epidermidis is an opportunistic pathogen commonly implicated in medical device-related infections. Its propensity to form biofilms not only leads to chronic infections but also exacerbates the issue of antibiotic resistance, necessitating high-dose antimicrobial treatments. In this study, we explored the use of diclofenac sodium, a non-steroidal anti-inflammatory drug, as an anti-biofilm agent against S. epidermidis. In this study, crystal violet staining and confocal laser scanning microscope analysis showed that diclofenac sodium, at subinhibitory concentration (0.4 mM), significantly inhibited biofilm formation in both methicillin-susceptible and methicillin-resistant S. epidermidis isolates. MTT assays demonstrated that 0.4 mM diclofenac sodium reduced the metabolic activity of biofilms by 25.21-49.01% compared to untreated controls. Additionally, the treatment of diclofenac sodium resulted in a significant decrease (56.01-65.67%) in initial bacterial adhesion, a crucial early phase of biofilm development. Notably, diclofenac sodium decreased the production of polysaccharide intercellular adhesin (PIA), a key component of the S. epidermidis biofilm matrix, in a dose-dependent manner. Real-time quantitative PCR analysis revealed that diclofenac sodium treatment downregulated biofilm-associated genes icaA, fnbA, and sigB and upregulated negative regulatory genes icaR and luxS, providing potential mechanistic insights. These findings indicate that diclofenac sodium inhibits S. epidermidis biofilm formation by affecting initial bacterial adhesion and the PIA synthesis. This underscores the potential of diclofenac sodium as a supplementary antimicrobial agent in combating staphylococcal biofilm-associated infections.

Antibiofilm Mechanisms of the Helical G3 Peptide against Staphylococcus epidermidis.

Antibacterial peptides (ABPs) have been recognized as promising alternatives to conventional antibiotics due to their broad antibacterial spectrum, high antibacterial activity, and low possibility of inducing bacterial resistance. However, their antibiofilm mechanisms have not yet reached a consensus. In this study, we investigated the antibiofilm activity of a short helical peptide G3 against Staphylococcus epidermidis, one of the most important strains of medical device contamination. Studies show that G3 inhibits S. epidermidis biofilm formation in a variety of ways. In the initial adhesion stage, G3 changes the properties of bacterial surfaces, such as charges, hydrophobicity, and permeability, by rapidly binding to them, thus interfering with their initial adhesion. In the mature stage, G3 prefers to target extracellular polysaccharides, leading to the death of outside bacteria and the disruption of the three-dimensional (3D) architecture of the bacterial biofilm. Such efficient antibiofilm activity of G3 endows it with great potential in the treatment of infections induced by the S. epidermidis biofilm.

Discovery of 1,2,3-triazole-based pleuromutilin derivatives as potent gram-positive antibacterial agents.

A novel class of pleuromutilin derivatives possessing 1,2,3-triazole as the linker connected to phenyl analogues were designed. The antibacterial properties of the prepared compounds were assessed in vitro against five strains (E. coli, S. aureus, S. epidermidis, and E. faecalis). Most of the tested compounds displayed potent antibacterial activities against gram-positive bacteria and 14-O-[2-(4-((2,4-dinitrophenoxy)-methyl-1H-1,2,3-triazol-1-yl) acetamide)-2-methylpropan-2-yl) thioacetyl]mutilin (7c) exerted antibacterial activities against S. aureus, MRSA and S. epidermidis with MIC values 0.0625 µg/mL, representing 64-fold, 4-fold and 8-fold higher than tiamulin respectively. Compound 6e, 7c and 8c were chosen to carry out killing kinetics, which exhibited concentration-dependent effect. Subsequently, molecular modeling was conducted to further explore the binding of compound 6e, 7a, 7c, 8c and tiamulin with 50S ribosomal subunit from deinococcus radiodurans. The investigation revealed that the main interactions between compound 7c and the ribosomal residues were three hydrogen bonds, π-π, and p-π conjugate effects. Additionally, the free binding energy and docking score of 7c with the ribosome demonstrated the lowest values of -11.90 kcal/mol and -7.97 kcal/mol, respectively, consistent with its superior antibacterial activities.

Genomic characterization of antibiotic-resistant Staphylococcus epidermidis with observed shifts in optimal temperature.

AIMS: Antibiotic resistance genes (ARGs) in the environment pose significant public health concerns and are influenced by conditions like temperature changes. We previously observed that resistance evolution to gentamicin and colistin affects optimal growth temperatures in Staphylococcus epidermidis isolates. Despite significant phenotype observations, the genetic basis remains unclear. We aim to identify the genetic changes linked to antibiotic resistance evolution that alter optimal growth temperature. METHODS AND RESULTS: Using whole-genome sequencing, we sequenced the genomes of gentamicin-resistant (GEN-1, GEN-2) and colistin-resistant (COL-4, COL-6) S. epidermidis isolates. Variant analysis with the BV-BRC bioinformatics tool identified genes involved in antibiotic resistance and temperature response. We found 12 genetic variants, including two unique to GEN-2 and one in COL-4. One shared mutation was observed in GEN-1 and GEN-2, and another in COL-4 and COL-6. Five mutations were shared among all isolates related to mobile gene elements, including a transposase IS4 family, two putative transposases, and two transposase-like insertion elements. CONCLUSIONS: Our findings indicate that the same genes involved in gentamicin and colistin resistance, especially those related to mobile genetic elements, may also play a crucial role in temperature response.

Dual-function antimicrobial-antibiofilm peptide hybrid to tackle biofilm-forming Staphylococcus epidermidis.

BACKGROUND: Due to their resistance and difficulty in treatment, biofilm-associated infections are problematic among hospitalized patients globally and account for 60% of all bacterial infections in humans. Antibiofilm peptides have recently emerged as an alternative treatment since they can be effectively designed and exert a different mode of biofilm inhibition and eradication. METHODS: A novel antibiofilm peptide, BiF, was designed from the conserved sequence of 18 α-helical antibiofilm peptides by template-assisted technique and its activity was improved by hybridization with a lipid binding motif (KILRR). Novel antibiofilm peptide derivatives were modified by substituting hydrophobic amino acids at positions 5 or 7, and both, with positively charged lysines (L5K, L7K). These peptide derivatives were tested for antibiofilm and antimicrobial activities against biofilm-forming Staphylococcus epidermidis and multiple other microbes using crystal violet and broth microdilution assays, respectively. To assess their impact on mammalian cells, the toxicity of peptides was determined through hemolysis and cytotoxicity assays. The stability of candidate peptide, BiF2_5K7K, was assessed in human serum and its secondary structure in bacterial membrane-like environments was analyzed using circular dichroism. The action of BiF2_5K7K on planktonic S. epidermidis and its effect on biofilm cell viability were assessed via viable counting assays. Its biofilm inhibition mechanism was investigated through confocal laser scanning microscopy and transcription analysis. Additionally, its ability to eradicate mature biofilms was examined using colony counting. Finally, a preliminary evaluation involved coating a catheter with BiF2_5K7K to assess its preventive efficacy against S. epidermidis biofilm formation on the catheter and its surrounding area. RESULTS: BiF2_5K7K, the modified antibiofilm peptide, exhibited dose-dependent antibiofilm activity against S. epidermidis. It inhibited biofilm formation at subinhibitory concentrations by altering S. epidermidis extracellular polysaccharide production and quorum-sensing gene expression. Additionally, it exhibited broad-spectrum antimicrobial activity and no significant hemolysis or toxicity against mammalian cell lines was observed. Its activity is retained when exposed to human serum. In bacterial membrane-like environments, this peptide formed an α-helix amphipathic structure. Within 4 h, a reduction in the number of S. epidermidis colonies was observed, demonstrating the fast action of this peptide. As a preliminary test, a BiF2_5K7K-coated catheter was able to prevent the development of S. epidermidis biofilm both on the catheter surface and in its surrounding area. CONCLUSIONS: Due to the safety and effectiveness of BiF2_5K7K, we suggest that this peptide be further developed to combat biofilm infections, particularly those of biofilm-forming S. epidermidis.

Comparative analysis of linezolid, vancomycin, and hyperbaric oxygen therapies in a rat model of ventriculoperitoneal shunt infection.

PURPOSE: Staphylococcus epidermidis is the most common causative microorganism of ventriculoperitoneal shunt infections. This study aimed to compare linezolid and vancomycin treatments and to examine the effect of these antibiotics alone and combined with hyperbaric oxygen therapy on the amount of bacterial colonies in the experimental S. epidermidis shunt infection model. METHODS: A shunt catheter was placed in the cisterna magna of 49 adult male Wistar albino rats. The rats were randomly divided into seven groups, as follows: sterile control, infected control, vancomycin, linezolid, hyperbaric oxygen, vancomycin + hyperbaric oxygen, linezolid + hyperbaric oxygen. In all groups except the sterile control group, 0.2 ml 107 CFU/mL S. epidermidis was inoculated to the cisterna magna. Parenteral vancomycin was administered 40 mg/kg/day to the vancomycin groups, and 50 mg/kg/day of enteral linezolid to the linezolid groups. Hyperbaric oxygen groups were given 100% oxygen at a pressure of 2.4 ATA for 50 min a day. One day after the last treatment, colony quantities in the shunt catheters and CSF were analyzed. RESULTS: The number of CSF colonies in the linezolid group was significantly lower than in the vancomycin group (p < 0.05). The number of CSF colonies in the linezolid + HBO group was significantly lower than in the vancomycin + HBO group (p < 0.05). CONCLUSIONS: Linezolid treatment was found to be more effective than vancomycin in ventriculoperitoneal shunt infection caused by S. epidermidis. There was no statistical difference among other treatment groups. Hyperbaric oxygen therapy is shown to contribute to the sterilization of cultures.

The Marine Antimicrobial Peptide AOD with Intact Disulfide Bonds Has Remarkable Antibacterial and Anti-Biofilm Activity.

American Oyster Defensin (AOD) is a marine peptide that is derived from North American mussels. It has been demonstrated to exhibit potent antimicrobial activity and high safety in both in vitro and in vivo models. In this study, to facilitate synthesis, mutants of AOD with fewer disulfide bonds were designed and subjected to structural, antimicrobial, and anti-biofilm analysis. The antimicrobial activity of AOD-derived peptides decreased after reduction in the disulfide bond, and among its three derivatives, only AOD-1 inhibited very few bacteria with a MIC value of 64 µg/mL, whereas the others had no inhibitory effect on pathogenic bacteria. The findings demonstrated that full disulfide bonds are indispensable for bactericidal activity, with the α-helix playing a pivotal role in inhibiting bacterial membranes. Furthermore, the results of the ATP, ROS, membrane potential, and membrane fluidity assays demonstrated that intracellular ATP, reactive oxygen species, and membrane fluidity were all increased, while membrane potential was reduced. This indicated that AOD resulted in the impairment of membrane fluidity and induced metabolic disorders, ultimately leading to bacterial death. The inhibitory effect of AOD on the biofilm of S. epidermidis G-81 was determined through the crystal violet and confocal microscopy. The results demonstrated that AOD exhibited a notable inhibitory impact on the biofilm of S. epidermidis G-81. The minimum biofilm inhibitory concentration of AOD on S. epidermidis G-81 was 16 µg/mL, and the minimum biofilm scavenging concentration was 32 µg/mL, which exhibited superior efficacy compared to that of lincomycin. The inhibitory effect on the primary biofilm was 90.3%, and that on the mature biofilm was 82.85%, with a dose-dependent inhibition effect. Concurrently, AOD cleared intra-biofilm organisms and reduced the number of biofilm-holding bacteria by six orders of magnitude. These data indicate that disulfide bonds are essential to the structure and activity of AOD, and AOD may potentially become an effective dual-action antimicrobial and anti-biofilm agent.