Altered competitive fitness, antimicrobial susceptibility, and cellular morphology in a triclosan-induced small-colony variant of staphylococcus aureus

Staphylococcus aureus can produce small-colony variants (SCVs) that express various phenotypes. While their significance is unclear, SCV propagation may be influenced by relative fitness, antimicrobial susceptibility, and the underlying mechanism. We have investigated triclosan-induced generation of SCVs in six S. aureus strains, including methicillin-resistant S. aureus (MRSA). Parent strains (P0) were repeatedly passaged on concentration gradients of triclosan using a solid-state exposure system to generate P10. P10 was subsequently passaged without triclosan to generate X10. Susceptibility to triclosan and 7 antibiotics was assessed at all stages. For S. aureus ATCC 6538, SCVs were further characterized by determining microbicide susceptibility and competitive fitness. Cellular morphology was examined using electron microscopy, and protein expression was evaluated through proteomics. Triclosan susceptibility in all SCVs (which could be generated from 4/6 strains) was markedly decreased, while antibiotic susceptibility was significantly increased in the majority of cases. An SCV of S. aureus ATCC 6538 exhibited significantly increased susceptibility to all tested microbicides. Cross-wall formation was impaired in this bacterium, while expression of FabI, a target of triclosan, and IsaA, a lytic transglycosylase involved in cell division, was increased. The P10 SCV was 49% less fit than P0. In summary, triclosan exposure of S. aureus produced SCVs in 4/6 test bacteria, with decreased triclosan susceptibility but with generally increased antibiotic susceptibility. An SCV derived from S. aureus ATCC 6538 showed reduced competitive fitness, potentially due to impaired cell division. In this SCV, increased FabI expression could account for reduced triclosan susceptibility, while IsaA may be upregulated in response to cell division defects

Interpretación errónea del concepto de entropía

Background Cetylpyridinium chloride (CPC) and sodium fluoride augment oral hygiene by inactivating bacteria and inhibiting enamel demineralisation, respectively. However, there are few reports in the literature documenting the antibacterial efficacy of their combined use in mouthrinses. We have used six experimental systems to compare the antibacterial effects of mouthrinses containing 0.075 % CPC (test rinse, TR) or 0.075 % CPC with sodium fluoride (test fluoride rinse, TFR). Results Effects against planktonic bacteria were determined using viable counting (for Streptococcus mutans and salivary bacteria), a redox dye (for Actinomyces viscosus and salivary bacteria) and viable counting (for ex vivo oral rinses). Effects against saliva-derived biofilms were quantified using confocal microscopy and differential viable counting. Inhibition of biofilm formation was evaluated by pre-treating hydroxyapatite coupons with mouthrinses prior to inoculation. Otherwise-identical controls without CPC (control rinse and control fluoride rinse, CR and CFR, respectively), were included throughout. Compared to the controls, TFR and TR demonstrated significant antimicrobial effects in the redox assays, by viable counts (>3 log reductions) and in oral rinse samples (>1.25 log reductions, p 3 log difference, p < 0.05). Overall, there were no consistent differences in the activities of TR and TFR. Conclusions Sodium fluoride did not influence the antibacterial and anti-biofilm potency of CPC-containing formulations, supporting the combined use of CPC and sodium fluoride in mouthrinses to control oral bacteria and protect tooth enamel

Comparing the effectiveness of polymer debriding devices using a porcine wound biofilm model

Objective: Debridement to remove necrotic and/or infected tissue and promote active healing remains a cornerstone of contemporary chronic wound management. While there has been a recent shift toward less invasive polymer-based debriding devices, their efficacy requires rigorous evaluation.Approach: This study was designed to directly compare monofilament debriding devices to traditional gauze using a wounded porcine skin biofilm model with standardized application parameters. Biofilm removal was determined using a surface viability assay, bacterial counts, histological assessment, and scanning electron microscopy (SEM).Results: Quantitative analysis revealed that monofilament debriding devices outperformed the standard gauze, resulting in up to 100-fold greater reduction in bacterial counts. Interestingly, histological and morphological analyses suggested that debridement not only removed bacteria, but also differentially disrupted the bacterially-derived extracellular polymeric substance. Finally, SEM of post-debridement monofilaments showed structural changes in attached bacteria, implying a negative impact on viability.Innovation: This is the first study to combine controlled and defined debridement application with a biologically relevant ex vivo biofilm model to directly compare monofilament debriding devices.Conclusion: These data support the use of monofilament debriding devices for the removal of established wound biofilms and suggest variable efficacy towards biofilms composed of different species of bacteria

New data and the hard pomeron

New structure-function data are in excellent agreement with the existence of a hard pomeron, with intercept about 1.4. It gives a very economical description of the data. Having fixed 2 parameters from the data for the real-photon cross section $\sigma^{\gamma p}$, we need just 5 further parameters to fit the data for $F_2(x,Q^2)$ with $x\leq 0.001$. The available data range from $Q^2=0.045$ to 35 GeV$^2$. With guesses consistent with dimensional counting for the $x$ dependences of our three separate terms, the fit extends well to larger $x$ and to $Q^2=5000$ GeV$^2$. With no additional parameters, it gives a good description of data for the charm structure function $F_2^c(x,Q^2)$ from $Q^2=0$ to 130 GeV$^2$. The two pomerons also give a good description of both the $W$ and the $t$ dependence of $\gamma p\to J/\psi p$.Comment: 11 pages, plain tex, with 10 figures embedded using epsf. (Spurious figure removed.

Lactobacillus rhamnosus GG inhibits the toxic effects of Staphylococcus aureus on epidermal keratinocytes

Few studies have evaluated the potential benefits of the topical application of probiotic bacteria or material derived from them. We have investigated whether a probiotic bacterium, Lactobacillus rhamnosus GG, can inhibit Staphylococcus aureus infection of human primary keratinocytes in culture. When primary human keratinocytes were exposed to S. aureus, only 25% of the keratinocytes remained viable following 24 h of incubation. However, in the presence of 10(8) CFU/ml of live L. rhamnosus GG, the viability of the infected keratinocytes increased to 57% (P = 0.01). L. rhamnosus GG lysates and spent culture fluid also provided significant protection to keratinocytes, with 65% (P = 0.006) and 57% (P = 0.01) of cells, respectively, being viable following 24 h of incubation. Keratinocyte survival was significantly enhanced regardless of whether the probiotic was applied in the viable form or as cell lysates 2 h before or simultaneously with (P = 0.005) or 12 h after (P = 0.01) S. aureus infection. However, spent culture fluid was protective only if added before or simultaneously with S. aureus. With respect to mechanism, both L. rhamnosus GG lysate and spent culture fluid apparently inhibited adherence of S. aureus to keratinocytes by competitive exclusion, but only viable bacteria or the lysate could displace S. aureus (P = 0.04 and 0.01, respectively). Furthermore, growth of S. aureus was inhibited by either live bacteria or lysate but not spent culture fluid. Together, these data suggest at least two separate activities involved in the protective effects of L. rhamnosus GG against S. aureus, growth inhibition and reduction of bacterial adhesion

Does microbicide use in consumer products promote antimicrobial resistance? A critical review and recommendations for a cohesive approach to risk assessment

The increasing use of microbicides in consumer products is raising concerns related to enhanced microbicide resistance in bacteria and potential cross resistance to antibiotics. The recently published documents on this topic from the European Commission have spawned much interest to better understand the true extent of the putative links for the benefit of the manufacturers, regulators, and consumers alike. This white paper is based on a 2-day workshop (SEAC-Unilever, Bedford, United Kingdom; June 2012) in the fields of microbicide usage and resistance. It identifies gaps in our knowledge and also makes specific recommendations for harmonization of key terms and refinement/standardization of methods for testing microbicide resistance to better assess the impact and possible links with cross resistance to antibiotics. It also calls for a better cohesion in research in this field. Such information is crucial to developing any risk assessment framework on microbicide use notably in consumer products. The article also identifies key research questions where there are inadequate data, which, if addressed, could promote improved knowledge and understanding to assess any related risks for consumer and environmental safety

Loss of Function in Escherichia coli exposed to Environmentally Relevant Concentrations of Benzalkonium Chloride

Assessing the risk of resistance associated with biocide exposure commonly involves exposing microorganisms to biocides at concentrations close to the MIC. With the aim of representing exposure to environmental biocide residues, MG1655 was grown for 20 passages in the presence or absence of benzalkonium chloride (BAC) at 100 ng/L and 1000 ng/L (0.0002% and 0.002% of the MIC respectively). BAC susceptibility, planktonic growth rates, motility and biofilm-formation were assessed, and differentially expressed genes determined via RNA-sequencing. Planktonic growth rate and biofilm-formation were significantly reduced (p<0.001) following BAC adaptation, whilst BAC minimum bactericidal concentration increased two-fold. Transcriptomic analysis identified 289 upregulated and 391 downregulated genes after long-term BAC adaptation when compared to the respective control organism passaged in BAC-free-media. When the BAC-adapted bacterium was grown in biocide-free medium, 1052 genes were upregulated and 753 were down regulated. Repeated passage solely in biocide-free medium resulted in 460 upregulated and 476 downregulated genes compared to unexposed bacteria. Long-term exposure to environmentally relevant BAC concentrations increased the expression of genes associated with efflux and reduced gene expression associated with outer-membrane porins, motility and chemotaxis. This was manifested phenotypically through loss-of-function (motility). Repeated passage in a BAC-free-environment resulted in the up-regulation of multiple respiration-associated genes, which was reflected by increased growth rate. In summary, repeated exposure of to BAC residues resulted in significant alterations in global gene expression that were associated with minor decreases in biocide susceptibility, reductions in growth-rate and biofilm-formation, and loss of motility. Exposure to very low concentrations of biocide in the environment is a poorly understood risk factor for antimicrobial resistance. Repeated exposure to trace levels of the biocide BAC resulted in loss of function (motility) and a general reduction in bacterial fitness, but relatively minor decreases in susceptibility. These changes were accompanied by widespread changes in the transcriptome. This demonstrates the importance of including phenotypic characterisation in studies designed to assess the risks of biocide exposure. [Abstract copyright: Copyright © 2018 American Society for Microbiology.

Fatty acid supplementation reverses the small colony variant phenotype in triclosan-adapted staphylococcus aureus: Genetic, proteomic and phenotypic analyses

Staphylococcus aureus can develop a small colony variant (SCV) phenotype in response to sub-lethal exposure to the biocide triclosan. In the current study, whole genome sequencing was performed and changes in virulence were investigated in five Staphylococcus aureus strains following repeated exposure to triclosan. Following exposure, 4/5 formed SCV and exhibited point mutations in the triclosan target gene fabI with 2/4 SCVs showing mutations in both fabI and fabD. The SCV phenotype was in all cases immediately reversed by nutritional supplementation with fatty acids or by repeated growth in the absence of triclosan, although fabI mutations persisted in 3/4 reverted SCVs. Virulence, determined using keratinocyte invasion and Galleria mellonella pathogenicity assays was significantly (p < 0.05) attenuated in 3/4 SCVs and in the non-SCV triclosan-adapted bacterium. Proteomic analysis revealed elevated FabI in 2/3 SCV and down-regulation in a protein associated with virulence in 1/3 SCV. In summary, attenuated keratinocyte invasion and larval virulence in triclosan-induced SCVs was associated with decreases in growth rate and virulence factor expression. Mutation occurred in fabI, which encodes the main triclosan target in all SCVs and the phenotype was reversed by fatty acid supplementation, demonstrating an association between fatty acid metabolism and triclosan-induced SCV