H-NS is a novel transcriptional modulator of the ribonucleotide reductase genes in Escherichia coli.

Ribonucleotide reductases (RNRs) are essential enzymes for DNA synthesis because they are responsible for the production of the four deoxyribonucleotides (dNTPs) from their corresponding ribonucleotides. Escherichia coli contains two classes of aerobic RNRs, encoded by the nrdAB (class Ia) and nrdHIEF (class Ib) operons, and a third RNR class, which is functional under anaerobic conditions and is encoded by the nrdDG (class III) operon. Because cellular imbalances in the amounts of the four dNTPs cause an increase in the rate of mutagenesis, the activity and the expression of RNRs must be tightly regulated during bacterial chromosome replication. The transcriptional regulation of these genes requires several transcription factors (including DnaA, IciA, FIS [factor for inversion stimulation], Fnr, Fur, and NrdR), depending on the RNR class; however, the factors that dictate the expression of some RNR genes in response to different environmental conditions are not known. We show that H-NS modulates the expression of the nrdAB and nrdDG operons. H-NS represses expression both in aerobically and in anaerobically growing cells. Under aerobic conditions, repression occurs at the exponential phase of growth as well as at the transition from the exponential to the stationary phase, a period when no dNTPs are needed. Under anoxic conditions, repression occurs mainly in exponentially growing cells. Electrophoretic mobility assays performed with two DNA fragments from the regulatory region of the nrdAB operon demonstrated the direct interaction of H-NS with these sequences.

Mimicking the Intestinal Host-Pathogen Interactions in a 3D In Vitro Model: The Role of the Mucus Layer.

The intestinal mucus lines the luminal surface of the intestinal epithelium. This mucus is a dynamic semipermeable barrier and one of the first-line defense mechanisms against the outside environment, protecting the body against chemical, mechanical, or biological external insults. At the same time, the intestinal mucus accommodates the resident microbiota, providing nutrients and attachment sites, and therefore playing an essential role in the host-pathogen interactions and gut homeostasis. Underneath this mucus layer, the intestinal epithelium is organized into finger-like protrusions called villi and invaginations called crypts. This characteristic 3D architecture is known to influence the epithelial cell differentiation and function. However, when modelling in vitro the intestinal host-pathogen interactions, these two essential features, the intestinal mucus and the 3D topography are often not represented, thus limiting the relevance of the models. Here we present an in vitro model that mimics the small intestinal mucosa and its interactions with intestinal pathogens in a relevant manner, containing the secreted mucus layer and the epithelial barrier in a 3D villus-like hydrogel scaffold. This 3D architecture significantly enhanced the secretion of mucus. In infection with the pathogenic adherent invasive E. coli strain LF82, characteristic of Crohn's disease, we observed that this secreted mucus promoted the adhesion of the pathogen and at the same time had a protective effect upon its invasion. This pathogenic strain was able to survive inside the epithelial cells and trigger an inflammatory response that was milder when a thick mucus layer was present. Thus, we demonstrated that our model faithfully mimics the key features of the intestinal mucosa necessary to study the interactions with intestinal pathogens.

Development of a ternary cyclodextrin-arginine-ciprofloxacin antimicrobial complex with enhanced stability.

Designing useful functionalities in clinically validated, old antibiotics holds promise to provide the most economical solution for the global lack of effective antibiotics, as undoubtedly a serious health threat. Here we show that using the surface chemistry of the cyclodextrin (ßCD) cycle and arginine (arg) as a linker, provides more stable ternary antibiotic complex (ßCD-arg-cpx). In contrast to classical less stable inclusion complexes, which only modify antibiotic solubility, here-presented ternary complex is more stable and controls drug release. The components of the complex intensify interactions with bacterial membranes and increase the drug's availability inside bacterial cells, thereby improving its antimicrobial efficacy and safety profile. Multifunctional antibiotics, formulated as drug delivery systems per se, that take the drug to the site of action, maximize its efficacy, and provide optical detectability are envisaged as the future in fighting against infections. Their role as a tool against multiresistant strains remains as interesting challenge open for further research.

Pseudomonas aeruginosa biofilms and their partners in crime.

Pseudomonas aeruginosa biofilms and the capacity of the bacterium to coexist and interact with a broad range of microorganisms have a substantial clinical impact. This review focuses on the main traits of P. aeruginosa biofilms, such as the structural composition and regulatory networks involved, placing particular emphasis on the clinical challenges they represent in terms of antimicrobial susceptibility and biofilm infection clearance. Furthermore, the ability of P. aeruginosa to grow together with other microorganisms is a significant pathogenic attribute with clinical relevance; hence, the main microbial interactions of Pseudomonas are especially highlighted and detailed throughout this review. This article also explores the infections caused by single and polymicrobial biofilms of P. aeruginosa and the current models used to recreate them under laboratory conditions. Finally, the antimicrobial and antibiofilm strategies developed against P. aeruginosa mono and multispecies biofilms are detailed at the end of this review.

Nano-engineering stable contact-based antimicrobials: Chemistry at the interface between nano-gold and bacteria.

Contact-based antimicrobials, as antibiotic-free technologies that use non-specific interactions with bacterial cells to exert antimicrobial activity, are a prospective solution in fighting the global issue of bacterial resistance. A very simplified approach to their design considers the direct bonding of cationic guanidine-containing amino acids to the surface of nano-gold carriers. The structure enables antimicrobial activity due to a high density of cationic surface charges. This opens a set of novel questions that are important for their effective engineering, particularly regarding (i) chemistry and events that take place at the interface between NPs and cells, (ii) the direct influence of a charge (and its change) on interactions with bacterial and mammalian cells, and (iii) the stability of structures (and their antimicrobial activity) in the presence of enzymes, which are addressed in this paper. Because of the ability of amino acid-functionalized nano-gold to retain structural and functional activity, even after exposure to a range of physicochemical stimuli, they provide an excellent nanotechnological platform for designing highly effective contact-based antimicrobials and their applications.

Easily applicable modifications to electroporation conditions improve the transformation efficiency rates for rough morphotypes of fast-growing mycobacteria.

Electroporation is the most widely used and efficient method to transform mycobacteria. Through this technique, fast- and slow-growing mycobacteria with smooth and rough morphotypes have been successfully transformed. However, transformation efficiencies differ widely between species and strains. In this study, the smooth and rough morphotypes of Mycobacteroides abscessus and Mycolicibacterium brumae were used to improve current electroporation procedures for fast-growing rough mycobacteria. The focus was on minimizing three well-known and challenging limitations: the mycobacterial restriction-modification systems, which degrade foreign DNA; clump formation of electrocompetent cells before electroporation; and electrical discharges during pulse delivery, which were reduced by using salt-free DNA solution. Herein, different strategies are presented that successfully address these three limitations and clearly improve the electroporation efficiencies over the current procedures. The results demonstrated that combining the developed strategies during electroporation is highly recommended for the transformation of fast-growing rough mycobacteria.

Utility of Galleria mellonella larvae for evaluating nanoparticle toxicology.

The use of nanoparticles in consumer products is currently on the rise, so it is important to have reliable methods to predict any associated toxicity effects. Traditional in vitro assays fail to mimic true physiological responses of living organisms against nanoparticles whereas murine in vivo models are costly and ethically controversial. For these reasons, this study aimed to evaluate the efficacy of Galleria mellonella as an alternative, non-rodent in vivo model for examining nanoparticle toxicity. Silver, selenium, and functionalized gold nanoparticles were synthesized, and their toxicity was assessed in G. mellonella larvae. The degree of acute toxicity effects caused by each type of NP was efficiently detected by an array of indicators within the larvae: LD50 calculation, hemocyte proliferation, NP distribution, behavioral changes, and histological alterations. G. mellonella larvae are proposed as a nanotoxicological model that can be used as a bridge between in vitro and in vivo murine assays in order to obtain better predictions of NP toxicity.

Morphological and cytokine profiles as key parameters to distinguish between Gram-negative and Gram-positive bacterial keratitis.

Bacterial keratitis (BK) is an ocular disorder associated with poor visual prognosis. Quantification of the associated inflammatory response may provide insight into the pathogenesis of BK and guide treatment options. In this exploratory study, we evaluated 45 BK patients and 20 healthy controls by optical coherence tomography and pro-inflammatory tear cytokine analysis. The aim was to quantify the differential morphological and cytokine inflammatory response between Gram-negative and Gram-positive BK and to determine the diagnostic value of corneal thickness (CT) and infiltrate thickness (IT) in distinguishing Gram-ve BK in a clinical cohort. Greater CT and IT, at clinical presentation, were indicative of Gram-ve infection with values detected of ≥ 950 µm and ≥ 450 µm, respectively. Combination of these CT and IT values had a 100% sensitivity and 83.3% specificity as a diagnostic indicator of Gram-ve infection. Similarly, there were higher levels of IL-1ß, IL-6 and IL-8 cytokines were quantified in keratitis caused by Gram-negative bacteria. Among the different tear cytokines analysed, a significant reduction after three days of treatment was detected for pro-inflammatory cytokines IL-1ß, IL-2, IL-6, IL-8 and TNF-α, prior to starting with the administration of steroid drops. Overall, this study shows the potential value of serial OCT and tear cytokine measurements in the management of BK.

Optimal environmental and culture conditions allow the in vitro coexistence of Pseudomonas aeruginosa and Staphylococcus aureus in stable biofilms.

The coexistence between species that occurs in some infections remains hard to achieve in vitro since bacterial fitness differences eventually lead to a single organism dominating the mixed culture. Pseudomonas aeruginosa and Staphylococcus aureus are major pathogens found growing together in biofilms in disease-affected lungs or wounds. Herein, we tested and analyzed different culture media, additives and environmental conditions to support P. aeruginosa and S. aureus coexistence in vitro. We have unraveled the potential of DMEM to support the growth of these two organisms in mature cocultured biofilms (three days old) in an environment that dampens the pH rise. Our conditions use equal initial inoculation ratios of both strains and allow the stable formation of separate S. aureus microcolonies that grow embedded in a P. aeruginosa biofilm, as well as S. aureus biofilm overgrowth when bovine serum albumin is added to the system. Remarkably, we also found that S. aureus survival is strictly dependent on a well-characterized phenomenon of oxygen stratification present in the coculture biofilm. An analysis of differential tolerance to gentamicin and ciprofloxacin treatment, depending on whether P. aeruginosa and S. aureus were growing in mono- or coculture biofilms, was used to validate our in vitro coculture conditions.

Signaling Mediated by Toll-Like Receptor 5 Sensing of Pseudomonas aeruginosa Flagellin Influences IL-1ß and IL-18 Production by Primary Fibroblasts Derived from the Human Cornea.

Pseudomonas aeruginosa is the principal cause of bacterial keratitis worldwide and overstimulation of the innate immune system by this organism is believed to contribute significantly to sight loss. In the current study, we have used primary human corneal fibroblast (hCF) cells as an ex vivo model of corneal infection to examine the role of P. aeruginosa flagellum and type three secretion system (TTSS) in inducing inflammasome-associated molecules that trigger IL-1ß and IL-18 production during the early stages of the infection. Our results show that P. aeruginosa infection stimulated the non-canonical pathway for IL-1ß and IL-18 expression and pathway stimulation was influenced predominantly by the flagellum. Both IL-1ß and IL-18 cytokines were expressed intracellularly during bacterial infection, but only the former was released and detected in the extracellular environment. We also investigated the signaling pathways in hCFs mediated by Toll-Like Receptor (TLR)4 and TLR5 sensing of P. aeruginosa, and our data show that the signal triggered by TLR5-flagellin sensing significantly contributed to IL-1ß and IL-18 cytokine production in our model. Our study suggests that IL-18 expression is wholly dependent on extracellular flagellin sensing by TLR5, whereas IL-1ß expression is also influenced by P. aeruginosa lipopolysacharide. Additionally, we demonstrate that IL-1ß and IL-18 production by hCFs can be triggered by both MyD88-dependent and -independent pathways. Overall, our study provides a rationale for the development of targeted therapies, by proposing an inhibition of flagellin-PRR-signaling interactions, in order to ameliorate the inflammatory response characteristic of P. aeruginosa keratitis.

Biofilm modifies expression of ribonucleotide reductase genes in Escherichia coli.

Ribonucleotide reductase (RNR) is an essential enzyme for all living organisms since is the responsible for the last step in the synthesis of the four deoxyribonucleotides (dNTPs) necessary for DNA replication and repair. In this work, we have investigated the expression of the three-RNR classes (Ia, Ib and III) during Escherichia coli biofilm formation. We show the temporal and spatial importance of class Ib and III RNRs during this process in two different E. coli wild-type strains, the commensal MG1655 and the enteropathogenic and virulent E2348/69, the prototype for the enteropathogenic E. coli (EPEC). We have established that class Ib RNR, so far considered cryptic, play and important role during biofilm formation. The implication of this RNR class under the specific growth conditions of biofilm formation is discussed.