Investigation of Novel pmrB and eptA Mutations in Isogenic Acinetobacter baumannii Isolates Associated with Colistin Resistance and Increased Virulence In Vivo.

Colistin resistance in Acinetobacter baumannii is of great concern and is a threat to human health. In this study, we investigate the mechanisms of colistin resistance in four isogenic pairs of A. baumannii isolates displaying an increase in colistin MICs. A mutation in pmrB was detected in each colistin-resistant isolate, three of which were novel (A28V, I232T, and ΔL9-G12). Increased expression of pmrC was shown by semi-quantitative reverse transcription-PCR (qRT-PCR) for three colistin-resistant isolates, and the addition of phosphoethanolamine (PEtN) to lipid A by PmrC was revealed by mass spectrometry. Interestingly, PEtN addition was also observed in some colistin-susceptible isolates, indicating that this resistance mechanism might be strain specific and that other factors could contribute to colistin resistance. Furthermore, the introduction of pmrAB carrying the short amino acid deletion ΔL9-G12 into a pmrAB knockout strain resulted in increased pmrC expression and lipid A modification, but colistin MICs remained unchanged, further supporting the strain specificity of this colistin resistance mechanism. Of note, a mutation in the pmrC homologue eptA and a point mutation in ISAba1 upstream of eptA were associated with colistin resistance and increased eptA expression, which is a hitherto undescribed resistance mechanism. Moreover, no cost of fitness was observed for colistin-resistant isolates, while the virulence of these isolates was increased in a Galleria mellonella infection model. Although the mutations in pmrB were associated with colistin resistance, PEtN addition appears not to be the sole factor leading to colistin resistance, indicating that the mechanism of colistin resistance is far more complex than previously suspected and is potentially strain specific.

Fiber Bragg Grating Dilatometry in Extreme Magnetic Field and Cryogenic Conditions.

In this work, we review single mode SiO2 fiber Bragg grating techniques for dilatometry studies of small single-crystalline samples in the extreme environments of very high, continuous, and pulsed magnetic fields of up to 150 T and at cryogenic temperatures down to <1 K. Distinct millimeter-long materials are measured as part of the technique development, including metallic, insulating, and radioactive compounds. Experimental strategies are discussed for the observation and analysis of the related thermal expansion and magnetostriction of materials, which can achieve a strain sensitivity (ΔL/L) as low as a few parts in one hundred million (≈10-8). The impact of experimental artifacts, such as those originating in the temperature dependence of the fiber's index of diffraction, light polarization rotation in magnetic fields, and reduced strain transfer from millimeter-long specimens, is analyzed quantitatively using analytic models available in the literature. We compare the experimental results with model predictions in the small-sample limit, and discuss the uncovered discrepancies.

Evaluation of an Immunochromatographic Lateral Flow Assay (OXA-48 K-SeT) for Rapid Detection of OXA-48-Like Carbapenemases in Enterobacteriaceae.

We evaluated an immunochromatographic lateral flow assay to detect OXA-48-like carbapenemases (OXA-48 K-SeT) in Enterobacteriaceae (n = 82). One hundred percent sensitivity and specificity were observed using bacteria recovered from both solid medium and spiked blood culture bottles, and the results were obtained in <10 min.

In Vitro Antibacterial Activity of Curcumin-Polymyxin B Combinations against Multidrug-Resistant Bacteria Associated with Traumatic Wound Infections.

Bacterial infections resulting from nonsurgical traumatic wounds can be life threatening, especially those caused by multidrug-resistant (MDR) bacteria with limited therapeutic options. The antimicrobial activity of polymyxin B (1) and curcumin (2) alone and in combination was determined versus MDR bacterial isolates associated with traumatic wound infections. Cytotoxicity assays for 1 and 2 were undertaken in keratinocyte cell lines. Minimum inhibitory concentrations of 1 were significantly reduced in the presence of 2 (3- to 10-fold reduction), with synergy observed. Time-kill assays showed the combinations produced bactericidal activity. Cytotoxicity assays indicate the toxicity of 2 was reduced in the presence of 1.

Colistin and Fusidic Acid, a Novel Potent Synergistic Combination for Treatment of Multidrug-Resistant Acinetobacter baumannii Infections.

The spread of multidrug-resistant Acinetobacter baumannii (MDRAB) has led to the renaissance of colistin (COL), often the only agent to which MDRAB remains susceptible. Effective therapy with COL is beset with problems due to unpredictable pharmacokinetics, toxicity, and the rapid selection of resistance. Here, we describe a potent synergistic interaction when COL was combined with fusidic acid (FD) against A. baumannii. Synergy in vitro was assessed against 11 MDRAB isolates using disc diffusion, checkerboard methodology (fractional inhibitory concentration index [FICI] of ≤ 0.5, susceptibility breakpoint index [SBPI] of >2), and time-kill methodology (≥2 log10 CFU/ml reduction). The ability of FD to limit the emergence of COL resistance was assessed in the presence and absence of each drug alone and in combination. Synergy was demonstrated against all strains, with an average FICI and SBPI of 0.064 and 78.85, respectively. In time-kill assays, COL-FD was synergistic and rapidly bactericidal, including against COL-resistant strains. Fusidic acid prevented the emergence of COL resistance, which was readily selected with COL alone. This is the first description of a novel COL-FD regimen for the treatment of MDRAB. The combination was effective at low concentrations, which should be therapeutically achievable while limiting toxicity. Further studies are warranted to determine the mechanism underlying the interaction and the suitability of COL-FD as an unorthodox therapy for the treatment of multidrug-resistant Gram-negative infections.

In Vitro Activity of Epigallocatechin Gallate and Quercetin Alone and in Combination versus Clinical Isolates of Methicillin-Resistant Staphylococcus aureus.

Topical infections can become life threatening in immunocompromised patients. However, fewer treatments are available as multi-drug-resistant bacteria become more common. The natural compounds epigallocatechin gallate (1) and quercetin (2) alone and in combination were tested as potential antimicrobial clinical therapies. Strong antimicrobial activity was produced by 1 alone against methicillin-resistant Staphylococcus aureus, and activity was significantly increased in the presence of 2. A synergistic interaction was observed between the two compounds. Kill kinetics indicate the combination is bactericidal over 24 h.

In vitro and in vivo activities of tigecycline-colistin combination therapies against carbapenem-resistant Enterobacteriaceae.

We assessed the activity of tigecycline (TGC) combined with colistin (COL) against carbapenem-resistant enterobacteria. Synergy occurred in vitro against the majority of isolates, with the exception of Serratia marcescens. In a simple animal model (Galleria mellonella), TGC-COL was superior (P < 0.01) in treating Escherichia coli, Klebsiella pneumoniae, and Enterobacter infections, including those with TGC-COL resistance. Clinical studies are needed to determine whether TGC-COL regimens may be a viable option.

In vitro activity of curcumin in combination with epigallocatechin gallate (EGCG) versus multidrug-resistant Acinetobacter baumannii.

BACKGROUND: Acinetobacter baumannii is an opportunistic human pathogen often associated with life-threatening infections in the immunocompromised and the critically ill. Strains are often multidrug-resistant (MDR) and due to the lack of new synthetic antimicrobials in development for treatment, attention is increasingly focused on natural compounds either as stand-alone or adjunctive agents. Curcumin (CCM) is a natural polyphenol found in turmeric and isolated from the plant, Curcuma longa. Curcumin has been found to possess many biological properties, including antibacterial activity. In this study the antimicrobial activity of CCM and synergistic effects with epigallocatechin gallate (EGCG) against multidrug-resistant strains of A. baumannii were investigated and assessed via checkerboard and time-kill assays. RESULTS: The MIC of CCM was >256 µg/mL against all strains of A. baumannii whilst those for EGCG ranged from 128-1024 µg/mL. In checkerboard studies synergy was observed against 5/9 isolates, with an additive effect noted in the remaining 4. The addition of EGCG reduced the MIC of CCM by 3- to 7-fold, with the greatest interaction resulting in a CCM MIC of 4 µg/mL. Time-kill curves indicated that a CCM-EGCG (1:8 and 1:4) combination was bactericidal with a 4 to 5-log reduction in viable counts after 24 h compared to the most effective polyphenol alone. CONCLUSIONS: This study demonstrates that despite little antibacterial activity alone, CCM activity is greatly enhanced in the presence of EGCG resulting in antibacterial activity against MDR A. baumannii. The combination may have a potential use in medicine as a topical agent to prevent or treat A. baumannii infections.

The manganese carbonyl complex [Mn(CO)3(tqa-κ3N)]Br: A novel antimicrobial agent with the potential to treat avian pathogenic Escherichia coli (APEC) infections.

The development of alternatives to antibiotics is essential for the treatment of animal infections and as a measure to reduce the selective pressure on antibiotics that are critical for human medicine. Metal complexes have been highlighted for their antimicrobial activity against several bacterial pathogens. In particular, manganese carbonyl complexes have shown efficacy against multidrug-resistant Gram-negative pathogens, and relatively low cytotoxicity against avian macrophages and in wax moth larval models. They are thus potential candidates for deployment against Avian Pathogenic Escherichia coli (APEC), the aetiological agent of avian colibacillosis, which results in severe animal welfare issues and financial losses worldwide. This study aimed to determine the efficacy of [Mn(CO)3(tqa-κ3N)]Br in Galleria mellonella and chick models of infection against APEC. The results demonstrated in vitro and in vivo antibacterial activity against all antibiotic-resistant APEC test isolates screened in the study.

Prevalence of gastrointestinal parasites and molecular identification of beta-tubulin mutations associated with benzimidazole resistance in Haemonchus contortus in goats from selected districts of Uganda.

Gastrointestinal parasites are among the most economically important pathogens of small ruminants causing serious economic losses and animal welfare problems for the livestock industry worldwide. The emergence of anthelmintic resistant H. contortus in small ruminants is a serious problem because it undermines effective helminth control and results in reduced productivity. Little is known about resistance to benzimidazoles (BZ) in Haemonchus in goats and sheep in Uganda. The objective of this study was to determine the prevalence of gastrointestinal parasites and to identify the presence of benzimidazole resistance associated mutations in the ß-tubulin isotype 1 gene of Haemonchus contortus in goats from selected districts of Uganda. A total of 200 goats from 10 districts of Uganda slaughtered at Kalerwe abattoir in Kampala were sampled for H. contortus adult worms. Faecal samples were also collected to detect other intestinal parasites. Faecal microscopy and analysis were performed using flotation and sedimentation techniques. DNA was extracted from adult worms and PCR and sequencing of the ITS- 2 region and ß-tubulin isotype 1 gene performed to identify H. contortus species and to determine the presence of mutations associated with anthelmintic resistance respectively. Faecal microscopy showed that the most prevalent intestinal parasites were coccidia (98%), strongyles (97.5%), Strongyloides (82%), Paramphistomum (74.5%), Moniezia (46%), Fasciola (1.5%) and Trichuris (1%). Most goats had a high intestinal burden of coccidia (≥ 5000 oocyst per gram) and strongyles (≥ 1000 egg per gram), 65% and 67.5%, respectively. The prevalence of H. contortus adult worms was 63% (126/200). Sequencing of the partial ß-tubulin isotype 1 gene of 54 Haemonchus contortus adult male isolates revealed the presence of mutations associated with anthelmintic resistance. The F200Y mutation was the most common mutation (13% of samples with good beta-tubulin sequences) followed by the E198A and E198K mutations, both found in 9% of sequenced samples. Mutation F167Y was not identified in any of the samples and there were no heterozygous individuals for any of the SNPS associated with BZ resistance that were identified in this study. These findings highlight the need for controlled use of anthelmintics especially benzimidazoles, to enable sustainable control of H. contortus in Uganda, and a need for further investigation to understand the resistance of other parasites identified in this study.

Micropyramid-patterned, oxygen-permeable bottomed dish for high density culture of pancreatic islets.

The need for maintaining cell-spheroid viability and function within high-density cultures is unmet for various clinical and experimental applications, including cell therapies. One immediate application is for transplantation of pancreatic islets, a clinically recognized treatment option to cure type 1 diabetes; islets are isolated from a donor for subsequent culture prior to transplantation. However, high seeding conditions cause unsolicited fusion of multiple spheroids, thereby limiting oxygen diffusion to induce hypoxic cell death. Here we introduce a culture dish incorporating a micropyramid-patterned surface to prevent the unsolicited fusion and oxygen-permeable bottom for optimal oxygen environment. A 400µm-thick, oxygen-permeable polydimethylsiloxane sheet topped with micropyramid pattern of 400µm-base and 200µm-height was fabricated to apply to the 24-well plate format. The micropyramid pattern separated the individual pancreatic islets to prevent the fusion of multiple islets. This platform supported the high oxygen demand of islets at high seeding density at 260 islet equivalents cm-2, a 2-3-fold higher seeding density compared to the conventional islet culture used in a preparation for the clinical islet transplantations, demonstrating improved islet morphology, metabolism and function in a 4 d-culture. Transplantation of these islets into immunodeficient diabetic mice exhibited significantly improved engraftment to achieve euglycemia compared to islets cultured in the conventional culture wells. Collectively, this simple design modification allows for high-density cultures of three-dimensional cell spheroids to improve the viability and function for an array of investigational and clinical replacement tissues.

Composite pressure cell for pulsed magnets.

Extreme pressures and high magnetic fields can affect materials in profound and fascinating ways. However, large pressures and fields are often mutually incompatible; the rapidly changing fields provided by pulsed magnets induce eddy currents in the metallic components used in conventional pressure cells, causing serious heating, forces, and vibration. Here, we report a diamond-anvil-cell made mainly out of insulating composites that minimizes inductive heating while retaining sufficient strength to apply pressures of up to 8 GPa. Any residual metallic component is made of low-conductivity metals and patterned to reduce eddy currents. The simple design enables rapid sample or pressure changes, desired by pulsed-magnetic-field-facility users. The pressure cell has been used in pulsed magnetic fields of up to 65 T with no noticeable heating at cryogenic temperatures. Several measurement techniques are possible inside the cell at temperatures as low as 500 mK.

Zidovudine in synergistic combination with fosfomycin: an in vitro and in vivo evaluation against multidrug-resistant Enterobacterales.

Multidrug-resistant (MDR) Enterobacterales are a priority health issue with few treatment options. Recently, fosfomycin has been reconsidered for MDR bacterial infections. Zidovudine, licensed for the treatment of human immunodeficiency virus (HIV), has unexploited antibacterial properties and has been considered for drug repurposing. The aim of this study was to assess the effect of the combination of fosfomycin plus zidovudine against clinical MDR Enterobacterales isolates. Minimum inhibitory concentration (MIC) determination and checkerboard assays for 36 MDR Enterobacterales strains were performed. In addition, fosfomycin-resistant strains were evaluated using time-kill assay and in an in vivo Galleria mellonella infection model. Zidovudine and fosfomycin MICs ranged between 0.06 to >64 mg/L and 0.125 to >512 mg/L, respectively. A synergistic effect [fractional inhibitory concentration index (FICI) ≤0.5] was observed in 25 isolates and no antagonistic effect was observed in the remaining isolates. For 7 of 8 fosfomycin-resistant strains (MIC > 32 mg/L), zidovudine combination was able to restore fosfomycin susceptibility. These results were confirmed by time-kill assays. Fosfomycin + zidovudine presented greater larval survival (20-50%) than monotherapy. Synergistic activity was observed for fosfomycin + zidovudine in 69.4% of the tested strains. In vivo experiments confirmed the enhanced effectiveness of the combination. The zidovudine concentrations tested here can be reached in human serum using the actual licensed dosage, therefore this combination deserves further clinical investigation.

Protocol for Past BP: a randomised controlled trial of different blood pressure targets for people with a history of stroke of transient ischaemic attack (TIA) in primary care.

BACKGROUND: Blood pressure (BP) lowering in people who have had a stroke or transient ischaemic attack (TIA) leads to reduced risk of further stroke. However, it is not clear what the target BP should be, since intensification of therapy may lead to additional adverse effects. PAST BP will determine whether more intensive BP targets can be achieved in a primary care setting, and whether more intensive therapy is associated with adverse effects on quality of life. METHODS/DESIGN: This is a randomised controlled trial (RCT) in patients with a past history of stroke or TIA. Patients will be randomised to two groups and will either have their blood pressure (BP) lowered intensively to a target of 130 mmHg systolic, (or by 10 mmHg if the baseline systolic pressure is between 125 and 140 mmHg) compared to a standard group where the BP will be reduced to a target of 140 mmHg systolic. Patients will be managed by their practice at 1-3 month intervals depending on level of BP and followed-up by the research team at six monthly intervals for 12 months.610 patients will be recruited from approximately 50 general practices. The following exclusion criteria will be applied: systolic BP <125 mmHg at baseline, 3 or more anti-hypertensive agents, orthostatic hypotension, diabetes mellitus with microalbuminuria or other condition requiring a lower treatment target or terminal illness.The primary outcome will be change in systolic BP over twelve months. Secondary outcomes include quality of life, adverse events and cardiovascular events.In-depth interviews with 30 patients and 20 health care practitioners will be undertaken to investigate patient and healthcare professionals understanding and views of BP management. DISCUSSION: The results of this trial will inform whether intensive blood pressure targets can be achieved in people who have had a stroke or TIA in primary care, and help determine whether or not further research is required before recommending such targets for this population. TRIAL REGISTRATION: ISRCTN29062286.

Signature of optimal doping in Hall-effect measurements on a high-temperature superconductor.

High-temperature superconductivity is achieved by doping copper oxide insulators with charge carriers. The density of carriers in conducting materials can be determined from measurements of the Hall voltage--the voltage transverse to the flow of the electrical current that is proportional to an applied magnetic field. In common metals, this proportionality (the Hall coefficient) is robustly temperature independent. This is in marked contrast to the behaviour seen in high-temperature superconductors when in the 'normal' (resistive) state; the departure from expected behaviour is a key signature of the unconventional nature of the normal state, the origin of which remains a central controversy in condensed matter physics. Here we report the evolution of the low-temperature Hall coefficient in the normal state as the carrier density is increased, from the onset of superconductivity and beyond (where superconductivity has been suppressed by a magnetic field). Surprisingly, the Hall coefficient does not vary monotonically with doping but rather exhibits a sharp change at the optimal doping level for superconductivity. This observation supports the idea that two competing ground states underlie the high-temperature superconducting phase.

Rhenium diboride's monocrystal elastic constants, 308 to 5 K.

The five independent moduli required to construct the complete monocrystal elastic modulus tensor of the hexagonal-symmetry superhard compound ReB(2) were measured from 308 to 5 K using resonant ultrasound spectroscopy on a special-texture polycrystal. This is possible because, confirmed by X-ray diffraction, the specimen measured was composed of grains with hexagonal axes parallel so that its polycrystal elastic response is identical to a monocrystal and because hexagonal-symmetry solids are elastically isotropic in the plane perpendicular to the hexagonal axis. Along the hexagonal (c) axis, C(33) (0) = 1021 GPa, nearly equal to C(11) of diamond, and consistent with the superhard properties. However, in the (softer) isotropic plane, C(11) (0) = 671 GPa, much lower than diamond. The changes of C(ij) with temperature are very small and smooth. The Debye temperature was computed to be 738 K, and using a high-temperature approximation, the Grüneisen parameter is γ = 1.7.

Antibacterial green tea catechins from a molecular perspective: mechanisms of action and structure-activity relationships.

This review summarizes the mechanisms of antibacterial action of green tea catechins, discussing the structure-activity relationship (SAR) studies for each mechanism. The antibacterial activity of green tea catechins results from a variety of mechanisms that can be broadly classified into the following groups: (1) inhibition of virulence factors (toxins and extracellular matrix); (2) cell wall and cell membrane disruption; (3) inhibition of intracellular enzymes; (4) oxidative stress; (5) DNA damage; and (6) iron chelation. These mechanisms operate simultaneously with relative importance differing among bacterial strains. In all SAR studies, the highest antibacterial activity is observed for galloylated compounds (EGCG, ECG, and theaflavin digallate). This observation, combined with numerous experimental and theoretical evidence, suggests that catechins share a common binding mode, characterized by the formation of hydrogen bonds and hydrophobic interactions with their target.

Manganese complex [Mn(CO)3(tpa-κ3N)]Br increases antibiotic sensitivity in multidrug resistant Streptococcus pneumoniae.

OBJECTIVES: The emergence of multidrug-resistance (MDR) in Streptococcus pneumoniae clones and non-vaccine serotypes necessitate the development of novel treatment strategies. This work aimed to determine the efficacy of the Mn complex [Mn(CO)3(tpa-κ3N)]Br against clinically important MDR strains of S. pneumoniae. METHODS: Twenty MDR clinicalS. pneumoniae strains were included in this study. Minimum inhibitory concentrations (MICs) of [Mn(CO)3(tpa-κ3N)]Br were determined via broth microdilution alone and in combination with other antimicrobial agents using checkerboard assays and/or disc diffusion tests. In vitro efficacy was assessed by time-kill assays while in vivo efficacy was tested using the insect model Galleria mellonella. RESULTS: [Mn(CO)3(tpa-κ3N)]Br showed moderate in vitro efficacy against S. pneumoniae coupled with bactericidal activity. Checkerboard and disc diffusion assays showed synergy between [Mn(CO)3(tpa-κ3N)]Br and tetracycline, and the combination of both agents caused rapid kill-kinetics and reduced the MIC below the susceptibility breakpoint of 1 mg/L even for tetracycline-resistant strains of S. pneumoniae. Similar results were observed for the erythromycin- and the co-trimoxazole-Mn complex combination. In the G. mellonella infection model, mortality and morbidity rates at 96 h were significantly lower in larvae treated with [Mn(CO)3(tpa-κ3N)]Br than phosphate buffered saline, while treatment with the tetracycline-Mn complex combination was superior to monotherapy, resulting in significantly lower mortality and morbidity rates (p < 0.049). CONCLUSIONS: We show that [Mn(CO)3(tpa-κ3N)]Br has in vitro and in vivo antibacterial activity against clinically relevant strains of S. pneumoniae and has the potential to be used in combination with currently available antibiotics to increase their effectiveness against MDR S. pneumoniae.

Non-monotonic pressure dependence of high-field nematicity and magnetism in CeRhIn5.

CeRhIn5 provides a textbook example of quantum criticality in a heavy fermion system: Pressure suppresses local-moment antiferromagnetic (AFM) order and induces superconductivity in a dome around the associated quantum critical point (QCP) near pc ≈ 23 kbar. Strong magnetic fields also suppress the AFM order at a field-induced QCP at Bc ≈ 50 T. In its vicinity, a nematic phase at B* ≈ 28 T characterized by a large in-plane resistivity anisotropy emerges. Here, we directly investigate the interrelation between these phenomena via magnetoresistivity measurements under high pressure. As pressure increases, the nematic transition shifts to higher fields, until it vanishes just below pc. While pressure suppresses magnetic order in zero field as pc is approached, we find magnetism to strengthen under strong magnetic fields due to suppression of the Kondo effect. We reveal a strongly non-mean-field-like phase diagram, much richer than the common local-moment description of CeRhIn5 would suggest.

Diversity of amino acid substitutions in PmrCAB associated with colistin resistance in clinical isolates of Acinetobacter baumannii.

This study aimed to investigate the mechanisms of colistin resistance in 64 Acinetobacter baumannii isolates obtained from patients with ventilator-associated pneumonia hospitalised in Greece, Italy and Spain. In total, 31 A. baumannii isolates were colistin-resistant. Several novel amino acid substitutions in PmrCAB were found in 27 colistin-resistant A. baumannii. Most substitutions were detected in PmrB, indicating the importance of the histidine kinase for colistin resistance. In two colistin-resistant isolates, 93 amino acid changes were observed in PmrCAB compared with A. baumannii ACICU, and homologous recombination across different clonal lineages was suggested. Analysis of gene expression revealed increased pmrC expression in isolates harbouring pmrCAB mutations. Complementation of A. baumannii ATCC 19606 and ATCC 17978 with a pmrAB variant revealed increased pmrC expression but unchanged colistin MICs, indicating additional unknown factors associated with colistin resistance. Moreover, a combination of PmrB and PmrC alterations was associated with very high colistin MICs, suggesting accumulation of mutations as the mechanism for high-level resistance. The pmrC homologue eptA was detected in 29 colistin-susceptible and 26 colistin-resistant isolates. ISAba1 was found upstream of eptA in eight colistin-susceptible and one colistin-resistant isolate and eptA was disrupted by ISAba125 in two colistin-resistant isolates. Whilst in most isolates an association of eptA with colistin resistance was excluded, in one isolate an amino acid substitution in EptA (R127L) combined with a point mutation in ISAba1 upstream of eptA contributed to elevated colistin MICs. This study helps to gain an insight into the diversity and complexity of colistin resistance in A. baumannii.