Temperature-regulated efflux pump/potassium antiporter system mediates resistance to cationic antimicrobial peptides in Yersinia

Most bacterial pathogens are resistant to cationic antimicrobial peptides (CAMPs) that are key components of the innate immunity of both vertebrates and invertebrates. In Gram-negative bacteria, the known CAMPs resistance mechanisms involve outer membrane (OM) modifications and specifically those in the lipopolysaccharide (LPS) molecule. Here we report, the characterization of a novel CAMPs resistance mechanism present in Yersinia that is dependent on an efflux pump/potassium antiporter system formed by the RosA and RosB proteins. The RosA/RosB system is activated by a temperature shift to 37 degrees C, but is also induced by the presence of the CAMPs, such as polymyxin B. This is the first report of a CAMPs resistance system that is induced by the presence of CAMPs. It is proposed that the RosA/RosB system protects the bacteria by both acidifying the cytoplasm to prevent the CAMPs action and pumping the CAMPs out of the cell.

Brucella abortus and its closest phylogenetic relative, Ochrobactrum spp., differ in outer membrane permeability and cationic peptide resistance

The outer membrane (OM) of the intracellular parasite Brucella abortus is permeable to hydrophobic probes and resistant to destabilization by polycationic peptides and EDTA. The significance of these unusual properties was investigated in a comparative study with the opportunistic pathogens of the genus Ochrobactrum, the closest known Brucella relative. Ochrobactrum spp. OMs were impermeable to hydrophobic probes and sensitive to polymyxin B but resistant to EDTA. These properties were traced to lipopolysaccharide (LPS) because (i) insertion of B. abortus LPS, but not of Escherichia coli LPS, into Ochrobactrum OM increased its permeability; (ii) permeability and polymyxin B binding measured with LPS aggregates paralleled the results with live bacteria; and (iii) the predicted intermediate results were obtained with B. abortus-Ochrobactrum anthropi and E. coli-O. anthropi LPS hybrid aggregates. Although Ochrobactrum was sensitive to polymyxin, self-promoted uptake and bacterial lysis occurred without OM morphological changes, suggesting an unusual OM structural rigidity. Ochrobactrum and B. abortus LPSs showed no differences in phosphate, qualitative fatty acid composition, or acyl chain fluidity. However, Ochrobactrum LPS, but not B. abortus LPS, contained galacturonic acid. B. abortus and Ochrobactrum smooth LPS aggregates had similar size and zeta potential (-12 to -15 mV). Upon saturation with polymyxin, zeta potential became positive (1 mV) for Ochrobactrum smooth LPS while remaining negative (-5 mV) for B. abortus smooth LPS, suggesting hindered access to inner targets. These results show that although Ochrobactrum and Brucella share a basic OM pattern, subtle modifications in LPS core cause markedly different OM properties, possibly reflecting the adaptive evolution of B. abortus to pathogenicity.

Bactericidal activity of Lys49 and Asp49 myotoxic phospholipases A2 from Bothrops asper snake venom--synthetic Lys49 myotoxin II-(115-129)-peptide identifies its bactericidal region

Mammalian group-II phospholipases A2 (PLA2) of inflammatory fluids display bactericidal properties, which are dependent on their enzymatic activity. This study shows that myotoxins II (Lys49) and III (Asp49), two group-II PLA2 isoforms from the venom of Bothrops asper, are lethal to a broad spectrum of bacteria. Since the catalytically inactive Lys49 myotoxin II isoform has similar bactericidal effects to its catalytically active Asp49 counterpart, a bactericidal mechanism that is independent of an intrinsic PLA2 activity is demonstrated. Moreover, a synthetic 13-residue peptide of myotoxin II, comprising residues 115-129 (common numbering system) near the C-terminal loop, reproduced the bactericidal effect of the intact protein. Following exposure to the peptide or the protein, accelerated uptake of the hydrophobic probe N-phenyl-N-naphthylamine was observed in susceptible but not in resistant bacteria, indicating that the lethal effect was initiated on the bacterial membrane. The outer membrane, isolated lipopolysaccharide (LPS), and lipid A of susceptible bacteria showed higher binding to the myotoxin II-(115-129)-peptide than the corresponding moieties of resistant strains. Bacterial LPS chimeras indicated that LPS is a relevant target for myotoxin II-(115-129)-peptide. When heterologous LPS of the resistant strain was present in the context of susceptible bacteria, the chimera became resistant, and vice versa. Myotoxin II represents a group-II PLA2 with a direct bactericidal effect that is independent of an intrinsic enzymatic activity, but adscribed to the presence of a short cluster of basic/hydrophobic amino acids near its C-terminal loop.

Effect of a multifaceted intervention on number of antimicrobial prescriptions for suspected urinary tract infections in residents of nursing homes:Cluster randomised controlled trial

Objective: To assess whether a multifaceted intervention can reduce the number of prescriptions for antimicrobials for suspected urinary tract infections in residents of nursing homes. Design: Cluster randomised controlled trial. Setting: 24 nursing homes in Ontario, Canada, and Idaho, United States. Participants: 12 nursing homes allocated to a multifaceted intervention and 12 allocated to usual care. Outcomes were measured in 4217 residents. Interventions: Diagnostic and treatment algorithm for urinary tract infections implemented at the nursing home level using a multifaceted approach-small group interactive sessions for nurses, videotapes, written material, outreach visits, and one on one interviews with physicians. Main outcome measures: Number of antimicrobials prescribed for suspected urinary tract infections, total use of antimicrobials, admissions to hospital, and deaths. Results: Fewer courses of antimicrobials for suspected urinary tract infections per 1000 resident days were prescribed in the intervention nursing homes than in the usual care homes (1.17 v 1.59 courses; weighted mean difference -0.49, 95% confidence intervals -0.93 to -0.06). Antimicrobials for suspected urinary tract infection represented 28.4% of all courses of drugs prescribed in the intervention nursing homes compared with 38.6% prescribed in the usual care homes (weighted mean difference -9.6%, -16.9% to -2.4%). The difference in total antimicrobial use per 1000 resident days between intervention and usual care groups was not significantly different (3.52 v 3.93; weighed mean difference -0.37, -1.17 to 0.44). No significant difference was found in admissions to hospital or mortality between the study arms. Conclusion: A multifaceted intervention using algorithms can reduce the number of antimicrobial prescriptions for suspected urinary tract infections in residents of nursing homes.

Enhanced antimicrobial activities of 1-alkyl-3-methyl imidazolium ionic liquids based on silver or copper containing anions

We have developed a series of 1-alkyl-3-methylimidazolium tetrachlorocuprate(II) and dibromoargentate(I) ionic liquids with enhanced antimicrobial activity when compared with 1-alkyl-3-methylimidazolium chloride ionic liquids. These new ionic liquids proved to be effective against a range of pathogenic bacteria and fungi.

Targeting treatment resistant breast cancer stem cells with FKBPL and its peptide derivative, AD-01, via the CD44 pathway

PURPOSE: FKBPL and its peptide derivative, AD-01, have already demonstrated tumour growth inhibition and CD44 dependent anti-angiogenic activity. Here we explore the ability of AD-01 to target CD44 positive breast cancer stem cells (BCSCs). EXPERIMENTAL DESIGN: Mammosphere assays and flow cytometry were utilized to analyse the effect of FKBPL overexpression/knockdown and AD-01 treatment ± other anti-cancer agents on BCSCs using breast cancer cell lines (MCF-7/MDA-231/ZR-75), primary patient samples and xenografts. Delays in tumour initiation were evaluated in vivo. The anti-stem cell mechanisms were determined using clonogenic assays, qPCR and immunofluorescence. RESULTS: AD-01 treatment was highly effective at inhibiting the BCSC population by reducing mammosphere forming efficiency (MFE) and ESA+/CD44+/CD24- or ALDH+ cell subpopulations in vitro and tumour initiation in vivo. The ability of AD-01 to inhibit the self-renewal capacity of BCSCs was confirmed; mammospheres were completely eradicated by the third generation. The mechanism appears to be due to AD-01-mediated BCSC differentiation demonstrated by a significant decrease in the number of holoclones and an associated increase in meroclones/paraclones; the stem cell markers, Nanog, Oct4 and Sox2, were also significantly reduced. Furthermore, we demonstrated additive inhibitory effects when AD-01 was combined with the Notch inhibitor, DAPT. AD-01 was also able to abrogate a chemo- and radiotherapy induced enrichment in BCSCs. Finally, FKBPL knockdown led to an increase in Nanog/Oct4/Sox2 and an increase in BCSCs, highlighting a role for endogenous FKBPL in stem cell signalling. CONCLUSIONS: AD-01 has dual anti-angiogenic and anti-BCSC activity which will be advantageous as this agent enters clinical trial.

Efficacy of Biocides Used in the Modern Food Industry To Control Salmonella enterica, and Links between Biocide Tolerance and Resistance to Clinically Relevant Antimicrobial Compounds

Biocides play an essential role in limiting the spread of infectious disease. The food industry is dependent on these agents, and their increasing use is a matter for concern. Specifically, the emergence of bacteria demonstrating increased tolerance to biocides, coupled with the potential for the development of a phenotype of cross-resistance to clinically important antimicrobial compounds, needs to be assessed. In this study, we investigated the tolerance of a collection of susceptible and multidrug-resistant (MDR) Salmonella enterica strains to a panel of seven commercially available food-grade biocide formulations. We explored their abilities to adapt to these formulations and their active biocidal agents, i.e., triclosan, chlorhexidine, hydrogen peroxide, and benzalkonium chloride, after sequential rounds of in vitro selection. Finally, cross-tolerance of different categories of biocidal formulations, their active agents, and the potential for coselection of resistance to clinically important antibiotics were investigated. Six of seven food-grade biocide formulations were bactericidal at their recommended working concentrations. All showed a reduced activity against both surface-dried and biofilm cultures. A stable phenotype of tolerance to biocide formulations could not be selected. Upon exposure of Salmonella strains to an active biocidal compound, a high-level of tolerance was selected for a number of Salmonella serotypes. No cross-tolerance to the different biocidal agents or food-grade biocide formulations was observed. Most tolerant isolates displayed changes in their patterns of susceptibility to antimicrobial compounds. Food industry biocides are effective against planktonic Salmonella. When exposed to sublethal concentrations of individual active biocidal agents, tolerant isolates may emerge. This emergence was associated with changes in antimicrobial susceptibilities.

The future of antimicrobial therapy in the era of antibiotic resistance in cystic fibrosis pulmonary infection

Cystic fibrosis (CF) is characterised by chronic polymicrobial airway infection and inflammation, which is the major cause of morbidity and mortality. Aggressive use of antimicrobials has been fundamental in increasing the life expectancy of CF patients in recent years. However, enhanced culture and non-culture based detection methods have identified bacteria in the CF lung not previously isolated from CF patients by routine diagnostic microbiology Coupled with increasing antimicrobial resistance, the future of antimicrobial therapy in CF respiratory infection remains challenging. New strategies are needed to address these problems and ensure improvements in life expectancy are maintained. Potential future strategies include the use of new antimicrobial agents and formulations currently in clinical trials, alternative methods of selecting appropriate therapeutic regimens, determination of the pathogenicity of species newly associated with CF and the development of new antimicrobials and adjuvants for use in clinical practice.

WO 2014087023 An Amphipathic Peptide

http://www.google.com/patents/EP2928909A1?cl=en

Hydrogel-forming microneedle arrays exhibit antimicrobial properties:potential for enhanced patient safety

We describe, for the first time, the microbial characterisation of hydrogel-forming polymeric microneedle arrays and the potential for passage of microorganisms into skin following microneedle penetration. Uniquely, we also present insights into the storage stability of these hydroscopic formulations, from physical and microbiological viewpoints, and examine clinical performance and safety in human volunteers. Experiments employing excised porcine skin and radiolabelled microorganisms showed that microorganisms can penetrate skin beyond the stratum corneum following microneedle puncture. Indeed, the numbers of microorganisms crossing the stratum corneum following microneedle puncture were greater than 105 cfu in each case. However, no microorganisms crossed the epidermal skin. When using a 21G hypodermic needle, more than 104 microorganisms penetrated into the viable tissue and 106 cfu of Candida albicans and Staphylococcus epidermidis completely crossed the epidermal skin in 24 h. The hydrogel-forming materials contained no microorganisms following de-moulding and exhibited no microbial growth during storage, while also maintaining their mechanical strength, apart from when stored at relative humidities of 86%. No microbial penetration through the swelling microneedles was detectable, while human volunteer studies confirmed that skin or systemic infection is highly unlikely when polymeric microneedles are used for transdermal drug delivery. Since no pharmacopoeial standards currently exist for microneedle-based products, the exact requirements for a proprietary product based on hydrogel-forming microneedles are at present unclear. However, we are currently working towards a comprehensive specification set for this microneedle system that may inform future developments in this regard.