Double Resistance Research Articles

A hybrid Genetic Algorithm and Levenberg-Marquardt (GA-LM) method for cell suspension measurement with electrical impedance spectroscopy.

A hybrid Genetic Algorithm (GA) and Levenberg-Marquardt (GA-LM) method is proposed for cell suspension measurement with electrical impedance spectroscopy. This algorithm combines the GA with global search ability and Levenberg-Marquardt (LM) algorithm with local search ability, which has the advantages of high accuracy and high robustness. First, GA-LM is compared with GA and LM algorithm separately by ideal simulation. Second, Gaussian noise is added to the ideal simulation data. The anti-noise ability of the GA-LM is discussed. Finally, experiments are conducted to verify the practicability of the proposed GA-LM method. In the experiment, GA-LM is used to fit the impedance spectrum of yeast suspensions with different volume fractions and active states. The results show that the GA-LM algorithm can converge to the real value that is set in the simulation under ideal numerical simulation conditions. In the simulation within 2% noise level, the mean relative error of the parameter solution is less than 4%, and the root mean square error of the fitting is less than 0.4. This method also performs well in fitting of the experimental data. In addition, the electric double layer resistance and cell membrane capacitance are selected as the main indicators for the identification of yeast suspension concentration and activity, respectively.

Extracellular Stimulation of a Target Neuron in Micro-Patterned Neuronal Circuits Using a Pair of Needle Electrodes

BACKGROUND: Electrical stimulation of neurons is essential to control neuronal activities. Thus far, direct stimulation by whole-cell patch-clamp methods [1] and extracellular stimulation using multi-electrode arrays (MEAs) [2] have been performed commonly. While the whole-cell patch-clamp method merits from the precise control of stimulation, the use of MEAs takes advantage of maintaining neuronal activity over a long period owing to its non-invasiveness. An optogenetic technique [3] has a targeting precision of a single-cell level, but this has difficulties in applying fast stimulation patterns due to the slow photoresponse of opsin [4]. Alternative non-invasive methods capable of targeting a single neuron with arbitrary stimulation patterns open new possibility of controlling and monitoring neuronal activities over a long duration.In this presentation, we propose a novel method to combine cell-patterning techniques [5] with electrical stimulation of a target cell with a pair of needle electrodes [6]. We show that the firing of a single neuron cultured on the micropattern can be controlled by the proposed method. We also show the controllability of the stimulation by analogue circuit simulation of a neuron stimulated electrically by a pair of needle electrodes via culture medium.EXPERIMENTAL METHODS: Neurons were cultured on a micropatterned glass substrates in isolation. A pair of tungsten needles except for their tip apexes were coated with an insulating polymer and mounted on manipulators. Two needles were immersed in the medium so that the target neuron is placed in-between. The distance from the neuron to the needle tip was controlled to be several tens to 100 μm. A bipolar square pulse was applied between electrodes, and the neuronal activity was visualized by Ca imaging.SIMULATION: The membrane potential response of a neuron to extracellular stimulation was analysed using LTspice simulator [7]. The circuit model is shown in Fig. 1a. The integrate-and-fire neuron was connected electrically with two needle electrodes via the electric double layers and solution resistances. The key of the circuit simulation is to divide the neuron into two compartments, each is stimulated with either of two needle electrodes. We conjecture that the neuron fires if the membrane potential either of two compartments exceeds the threshold upon the electrical stimulation. We investigated the membrane potential response when the resistance corresponding to the relative distance from the neuron to the electrode, the capacitive component of the needles, and the shape of voltage input pulses were changed.RESULTS AND DISSCUSSION: As shown in Fig. 1b, the neuron fires if the amplitude of the bipolar square pulse exceeds 5 Vp-p. The amplitude required for the neuron firing decreases with increasing distance from the neuron to the needle electrodes. The LTspice simulation provided the following results: (i) The positive electrode hyperpolarizes the membrane potential, whereas the negative electrode depolarizes it, indicating the membrane potential of two compartments changes to inverse direction even if a bipolar pulse was applied, as shown in Fig. 1c. (ii) The closer the electrode is to the cell, the more the membrane potential of the neuron changes and the voltage drop in the solution resistance is comparatively small. (iii) The main factor of neuronal firing is the transient potential fluctuation caused by the capacitive component of the electrical double layer and the neuronal membrane. (iv) The membrane potential of both the compartments is sensitive to transient change of tip voltage and does not change if the system reaches the steady state. This is also true if the DC voltage is continuously applied. These results indicate that applying a voltage pulse of appropriate amplitude can stimulate only cells in the vicinity of needle electrodes. Only a single neuron in neuronal culture can be stimulated by the needle electrodes by placing the target neuron between two needles. The non-invasive stimulation can also be used for long-term stimulation and monitoring.[1] E. Neher, et al., Nature 260(1976) 799.[2] C.A. Thomas Jr., et al., Exp. Cell Res. 74(1972) 61.[3] K. Deisseroth, et al., J. Neurosci. 26(2006) 10380.[4] B.D. Allen, et al., Learn. Mem. 22(2015) 232.[5] L. Bekar, et al., Nat. Med. 14(2008) 75.[6] H. Yamamoto, et al., Appl. Phys. Lett. 113(2018) 133703.[7] https://www.analog.com/en/design-center/design-tools-and-calculators/ltspice-simulator.html# Figure 1

(Keynote) EIS Study of Galvanic Corrosion of Al Alloy Coupled with High Strength Steel in Simulated Atmospheres

Introduction Weight reduction of automobiles is required to improve fuel efficiency. Therefore, the uses of lightweight materials such as Al alloys and CFRP is expected to increase. When an automobile is made of multi-materials, there is a concern that galvanic corrosion and crevice corrosion may occur at the joint parts.We successfully applied EIS to study corrosion of metals covered with thin solution films [1–4]. A two-electrode cell, which was composed of two identical metal plates embedded in epoxy resin with a small gap distance, was used to measure the EIS. Under very thin solution films, uneven current distribution must be considered in the EIS measurements because the current lines may concentrate on the nearest edge of the two electrodes when AC voltage is applied. The EIS characteristics of a metal/solution film interface was successfully described by an equivalent circuit of transmission line (TML) [2,3]. We reported that accurate charge transfer resistance can be obtained by curve fitting the measured EIS with a TML circuit, even if the current lines are not evenly distributed on the electrode surface [1].In this study, EIS was applied to the study of galvanic corrosion of Al alloy coupled with high strength steel under thin NaCl solution films with different thicknesses. The monitoring of the galvanic corrosion of All alloy was also carried out under a wet-dry cyclic condition by EIS. Experimental Al alloy (6016–T4, 10 mm×10 mm) and high-strength steel (980 MPa, 10 mm×10 mm) were used as specimens. They were embedded in the epoxy resin at a gap distance of 0.1 mm. A solution film of 2 M NaCl was placed on the cell surface. Three different films thickness, 0.025, 0.050 and 0.1 mm were used. The EIS was measured under the condition that the potential difference of Al alloy and high strength steel was set to zero by potentiostat. Amplitude of AC voltage applied between the two electrodes was 10 mV and the frequency range was 10 kHz to 10 mHz. Using the same cell, the galvanic corrosion under wet-dry cyclic conditions was also monitored by continuously measuring two impedance at high and low frequency. To investigate the effect of crevice, the cells with and without a crevice were employed. Result and Discussion All Nyquist plots of the galvanic couple measured under thin solution films showed two capacitive semi-circles. One semicircle with a smaller time constant can be expressed by a parallel combination of double layer capacitance Cdl and charge transfer resistance Rct for the All alloy. Another semicircle with a larger time constant can be given by Cdl / Rct for the high strength steel. The corrosion rate (CR) of the Al alloy estimated from the Rct increases with decreasing the solution film thickness. This tendency is consistent with that of the galvanic current between the two electrodes.The galvanic corrosion behavior under the wet-dry cycles in the presence of chlorides was monitored. In above experiment, the optimum frequency for monitoring Rct of the Al alloy was determined to be 0.3 Hz. Using this frequency, the corrosion rate of the Al alloy coupled with the high strength steel was monitored under the wet-dry cycles. The corrosion rate of Al alloy changed in response to the wet-dry cycles. The galvanic corrosion behavior will be discussed in the conference on the basis of the EIS monitoring data. Acknowledgements This paper is based on results obtained from a project commissioned by the New Energy and Industrial Technology Development Organization (NEDO). References 1) Y. Shi, E. Tada, A. Nishikata, J. Electrochem. Soc. 162[4], C135-C139 (2015).2) A. Nishikata, Y. Ichihara, T. Tsuru, Corros. Sci. 37, 897-911(1995)3) A. Nishikata, Y. Ichihara, Y. Hayashi, T. Tsuru, J. Electrochem. Soc.144, 1244-1252 (1997)4) A. Nishikata, Q. Zhu, E. Tada, Corros. Sci. 87, 80-87 (2014)

THE VALUE OF BASAL EXPRESSION LEVEL OF HEMOXYGENASE-1 FOR SENSITIVITY OF HUMAN MELANOMA CELLS TO OXIDATIVE STRESS IN VITRO

Introduction.The molecular basis of radio- and photodynamic therapy (PDT), the mechanism of action of a number of antitumor chemotherapy drugs is oxidative stress (OS). The enzyme hemoxygenase-I (НO-1), a molecular marker of OS, is a key participant in the system of protection and adaptation of tumor cells under stress.Objective.To find out whether the sensitivity of human melanoma tumor cells to OS depends on the basal and modulator-induced levels of НO-1 expressionMaterial and methods.Human melanoma cell lines were used in the study. The expression of mRNA НO-1 in cells was studied by real-time RT-PCR, the reactive oxygen species content in cells – by flow cytometry and the cytotoxicity of drugs – by MTT assay.Results.According to our data, human melanoma cells have different basal levels of HO-1 transcription: high (3.0–3.5 o. u.) in lines MelIL, MelP, medium (1.5 o. u.) in lines MeWo, MelZ, MelIbr and low (0.5 o. e.) – MelMe, A375).There is no direct correlation between the level of basal cell expression of HO-1 and their sensitivity to the OS inducer – Н2О2. The hemin-induced increase in HO-1 expression in cells is accompanied by doubled resistance to Н2О2. It was found that HO-1 repression in the presence of apigenin was registered in melanoma cells with different basal levels, but sensitization to Н2О2 (2–4 times) was observed only for cells with medium (MeWo) and low (A375) levels of basal HO-1 expression. It was found that the decrease in basal expression of HO-1 induced by apigenin is accompanied by an increase in the reactive oxygen species content in cells.Conclusions.The results of our research allow us to recommend natural flavon apigenin, a modulator of HO-1 expression, for inclusion in the chemotherapy and PDT regimens to increase the effectiveness of human melanoma treatment.

Competition delays multi-drug resistance evolution during combination therapy

Combination therapies have shown remarkable success in preventing the evolution of resistance to multiple drugs, including HIV, tuberculosis, and cancer. Nevertheless, the rise in drug resistance still remains an important challenge. The capability to accurately predict the emergence of resistance, either to one or multiple drugs, may help to improve treatment options. Existing theoretical approaches often focus on exponential growth laws, which may not be realistic when scarce resources and competition limit growth. In this work, we study the emergence of single and double drug resistance in a model of combination therapy of two drugs. The model describes a sensitive strain, two types of single-resistant strains, and a double-resistant strain. We compare the probability that resistance emerges for three growth laws: exponential growth, logistic growth without competition between strains, and logistic growth with competition between strains. Using mathematical estimates and numerical simulations, we show that between-strain competition only affects the emergence of single resistance when resources are scarce. In contrast, the probability of double resistance is affected by between-strain competition over a wider space of resource availability. This indicates that competition between different resistant strains may be pertinent to identifying strategies for suppressing drug resistance, and that exponential models may overestimate the emergence of resistance to multiple drugs. A by-product of our work is an efficient strategy to evaluate probabilities of single and double resistance in models with multiple sequential mutations. This may be useful for a range of other problems in which the probability of resistance is of interest.

Negative Differential Resistance: Double Negative Differential Resistance Device Based on Hafnium Disulfide/Pentacene Hybrid Structure (Adv. Sci. 19/2020)

In article number 2000991, Jeong Ho Cho, Jin-Hong Park, and co-workers report a double negative differential resistance (D-NDR) characteristic of a hybrid 2D van der Waals (2D vdW)/organic tunneling device consisting of a hafnium disulfide/pentacene heterojunction and a 3D pentacene resistor. This D-NDR device technology based on a hybrid 2D vdW/organic heterostructure provides a scientific foundation for various circuit applications that require the NDR phenomenon.

Optimal dynamic empirical therapy in a health care facility: A Monte-Carlo look-ahead method

Background and objectives: Empirical antimicrobial prescription strategies have been proposed to counteract the selection of resistant pathogenic strains. The respective merits of such strategies have been debated. Rather than comparing a finite number of policies, we take an optimization approach and propose a solution to the problem of finding an empirical therapy policy in a health care facility that minimizes the cumulative infected patient-days over a given time horizon. Methods: We assume that the parameters of the model are known and that when the policy is implemented, all patients receive the same treatment at a given time. We model the emergence and spread of antimicrobial resistance at the population level with the stochastic version of a compartmental model. The model features two drugs and the possibility of double resistance. Our solution method is a rollout algorithm. Results: In our example, the optimal policy computed with this method allows to reduce the average cumulative infected patient-days over two years by 22% compared to the best standard therapy. Considering regularity constraints, we could derive a policy with a fixed period and a performance close to that of the optimal policy. The average cumulative infected patient-days over two years obtained with the optimal policy is 6% lower (significantly at the 95% threshold) than that obtained with the fixed period policy. Conclusion: Our results illustrate the performance of a highly flexible solution method that will contribute to the development of implementable empirical therapy policies.

Effect of Double Pulse Resistance Spot Welding Process on 15B22 Hot Stamped Boron Steel

Double pulse resistance spot welding process by applying a second step welding current is a new pathway to alter the mechanical properties for advanced high strength steels. Herein, the resistance spot welding (RSW) of hot stamped boron steel 15B22 by one-step and two-step welding with different welding currents is investigated. The results of the tensile–shear test, size of the weld nugget, hardness distribution, microstructure, and failure mode of different welding parameters are analyzed. The weldment of the two-step RSW with a higher heat input exhibits a lower tensile–shear load and lower fracture energy when the size of the weld nugget is large. The microstructural study reveals the appearance of a partially melted zone and sub-critical heat affected zone in the weldment where the fracture readily occurred. Thus, the two-step RSW process weakens the strength of the sample, which is attributed to the partial softening in the weldment due to the higher heat input.

Double Negative Differential Resistance Device Based on Hafnium Disulfide/Pentacene Hybrid Structure.

Recently, combinations of 2D van der Waals (2D vdW) materials and organic materials have attracted attention because they facilitate the formation of various heterojunctions with excellent interface quality owing to the absence of dangling bonds on their surface. In this work, a double negative differential resistance (D‐NDR) characteristic of a hybrid 2D vdW/organic tunneling device consisting of a hafnium disulfide/pentacene heterojunction and a 3D pentacene resistor is reported. This D‐NDR phenomenon is achieved by precisely controlling an NDR peak voltage with the pentacene resistor and then integrating two distinct NDR devices in parallel. Then, the operation of a controllable‐gain amplifier configured with the D‐NDR device and an n‐channel transistor is demonstrated using the Cadence Spectre simulation platform. The proposed D‐NDR device technology based on a hybrid 2D vdW/organic heterostructure provides a scientific foundation for various circuit applications that require the NDR phenomenon.

A new method for identifying fractures in tight sandstone of a gentle structural area using well logs

AbstractIn this paper, we conducted a systematic study on the development characteristics and logging prediction of fractures of the Upper Paleozoic tight gas sandstone reservoirs in the eastern Ordos Basin. A new fracture prediction method was proposed based on the variable scale fracture probabilistic model. The calculation steps of this method were as follows: First, the sensitivity of each conventional log series to the tight sandstone fractures was analyzed. It was found that acoustic time difference (AC), density (DEN), natural gamma ray (GR), and deep investigate double lateral resistivity (RD) have good log responses to fractures. Then, the ratio of the range (R) to the standard deviation (S) of the log data was obtained. Finally, the second derivatives of the log series AC, DEN, GR, and RD were calculated, and a new fracture index was constructed (integrated second derivative, FC) using the multiple regression method. The fracture recognition rates of this method are distributed in 64.3%‐85.7%. Therefore, the constructed integrated second derivative (FC) based on conventional logs is effective to quantitatively characterize the fracture development of tight sandstone.

Evaluation of multiplex ARMS-PCR for detection of Helicobacter pylori mutations conferring resistance to clarithromycin and levofloxacin

BackgroundHelicobacter pylori bacterium is a major cause of gastritis. With increasing use of antibiotics to treat infections, mutation resistant strains have emerged in most human populations. To effectively treat patients to help resolve infections, the clinician needs information on the antibiotic susceptibility profile of the infection. Therefore, a rapid and accurate test is required to provide this information. To address this issue, we designed and validated a real time multiplex ARMS-PCR assay for rapid detection of highly prevalent H. pylori clarithromycin and levofloxacin resistance mutations. The aim of the study was to evaluate the analytical and diagnostic sensitivity and specificity of ARMS-PCR, using direct Sanger sequencing of the known resistance mutations as the gold standard.ResultsIn preliminary studies using a defined number of plasmids with clarithromycin and levofloxacin resistance mutations, the analytical sensitivity of our ARMS-PCR assay was 50 plasmid copies, equating to around 50 bacterium in a gastric biopsy sample. In terms of specificity, the assay was highly specific for the targeted resistance mutations. The assay was also able to reliably and efficiently detect heteroresistance of clarithromycin and levofloxacin mutations, even at a disproportional ratio of 1:1000. From the analysis of 192 samples with suspected H. pylori infections, the diagnostic sensitivity and specificity of the assay was very high for detection of clarithromycin resistance (100% and 100%), levofloxacin resistance (98.04% and 95.04%) and clarithromycin and levofloxacin double resistance (100% and 96.91%). Amongst the 74 patients diagnosed antibiotic resistance bacteria, 23 (31.1%) had clarithromycin resistance, 21 (28.4%) had levofloxacin resistance and 30 (40.5%) had double resistance. From sample receipt to results, our single tube assay could be routinely completed in under 2 h.ConclusionsOur assay demonstrated high diagnostic sensitivity and specificity for detection of clarithromycin and levofloxacin resistant H. pylori. Based on proven accuracy, together with high efficiency, scalability and low cost, our assay has useful clinical utility for rapid diagnosis of clarithromycin and levofloxacin resistant H. pylori infections. Our assay results will provide patients with a clear diagnosis, enabling the treating clinician to administer the most effective antibiotic regimen to help the clear the infection.

Accurate Empirical Fractional-Order Electrical Models of Young and Old Dentines.

Electrical impedance spectroscopy (EIS) is a fast, non-invasive, and safe technique for bioimpedance measurement. In dental research, EIS has been used to detect tooth cracks and caries with higher accuracy than visual and radiographic methods. Recently, a study has been reported on effect of age on impedance measurements for two age groups by employing EIS. The aim of that study was to demonstrate the usefulness of fractional calculus in equivalent circuit modeling. In proposed double dispersion Cole impedance (C-C) models, both resistance and pseudo-capacitance values were found to be significantly different for both age groups. However, in our study, the first time it was found out that proposed models' total pseudo-capacitance values of both young and old dentines can be reduced by 34% and 7.5%, respectively, if recurrent electrical impedance model for n = 2 bifurcations to be used. Secondly, new empirical fractional-order electrical models of human tooth using the optimized Valsa network with EIA standard compliant RC values are reported that provide better understanding of the structure of dentine from resistance and capacitance point of view.

Radial Expansion Facilitates the Maintenance of Double Antibiotic Resistances.

Most microbes live in spatially confined subpopulations. Under spatial structure conditions, the efficacy of natural selection is often reduced (relative to homogeneous conditions) due to the increased importance of genetic drift and local competition. Additionally, under spatial structure conditions, the fittest genotype may not always be the one with better access to the heterogeneous distribution of nutrients. The effect of radial expansion may be particularly relevant for the elimination of antibiotic resistance mutations, as their dynamics within bacterial populations are strongly dependent on their growth rate. Here, we use Escherichia coli to systematically compare the allele frequency of streptomycin, rifampin, and fluoroquinolone single and double resistance mutants after 24 h of coexistence with a susceptible strain under radial expansion (local competition) and homogeneous (global competition) conditions. We show that there is a significant effect of structure on the maintenance of double resistances which is not observed for single resistances. Radial expansion also facilitates the persistence of double resistances when competing against their single counterparts. Importantly, we found that spatial structure reduces the rate of compensation of the double mutant RpsLK43T RpoBH526Y and that a strongly compensatory mutation in homogeneous conditions becomes deleterious under spatial structure conditions. Overall, our results unravel the importance of spatial structure for facilitating the maintenance and accumulation of multiple resistances over time and for determining the identity of compensatory mutations.

Measurement Drift in Electrochemical Impedance Spectroscopy

Electrochemical impedance spectroscopy (EIS) is a powerful method that can be used to passively monitor disruptions on a surface. Yet, reproducibility issues associated with repeated impedance measurements using EIS is known.1,2 Often reproducibility issues are associated with differences in the initial state of the surface or quantifiable measurement drift of the EIS response. Measurement drift is of specific concern because it can result in mistaken interpretations of data.We have been investigating the cause and potential reasons for the electrochemical drift that has been observed. We hypothesize that the charging state of the working electrode influences the observed impedance drift in EIS. We investigated multiple factors to look at the charged state of the working electrode, including removing the working electrode from the solution, reporting the open circuit potential, reporting the double layer capacitance and charge transfer resistance changes, varying the concentration of an electrochemical mediator, changing the ionic strength of the aqueous solution, and DC offset conditions. The run-to-run variation was controlled by waiting 10 min between each run over a period of 1 hour (6 runs in total). Total charge transfer resistance drift varied by over 500 Ohms in the worst conditions and as good as 5 Ohms at the best conditions over six runs, indicating improvements of 2 orders of magnitude. All experimental variable changes were run in triplicate to show experimental error and standard deviations of all runs. Taking these methods into account, the most reproducible model of repeated EIS impedance measurements will be discussed. Further, theories and data to indicate potential causes and solutions will be presented.Acknowledgements: The authors would like to acknowledge the financial support from NIH (P20 GM113131) NSF (CBET 1638896), and the Hamel Center of Undergraduate Research at the University of New Hampshire. The authors also would like to acknowledge the assistance of Dr. Halpern’s laboratory.

Electrochemical Aptasensor for Detection of Dopamine

This work presents a proof of concept of a novel, simple, and sensitive method of detection of dopamine, a neurotransmitter within the human brain. We propose a simple electrochemical method for the detection of dopamine using a dopamine-specific aptamer labeled with an electrochemically active ferrocene tag. Aptamers immobilized on the surface of gold screen-printed gold electrodes via thiol groups can change their secondary structure by wrapping around the target molecule. As a result, the ferrocene labels move closer to the electrode surface and subsequently increase the electron transfer. The cyclic voltammograms and impedance spectra recorded on electrodes in buffer solutions containing different concentration of dopamine showed, respectively, the increase in both the anodic and cathodic currents and decrease in the double layer resistance upon increasing the concentration of dopamine from 0.1 to 10 nM L−1. The high affinity of aptamer-dopamine binding (KD ≈ 5 nM) was found by the analysis of the binding kinetics. The occurrence of aptamer-dopamine binding was directly confirmed with spectroscopic ellipsometry measurements.

The Study on Thermal Analysis Method of PCB

This essay makes the reliable analysis and modelling on printed circuit boards(PCB) by software of Flotherm and get conclusions of three points through simulation analysis:1) there is an important impact on heat transfer of the distribution of copper foil on PCB, and the actual condition should be selected for different precision simulation modules; 2) the heating device with simple structure and small thermal resistance can be dealt with as the simple square; 3) in the case of knowing the internal thermal resistance, integrated chip adopts double thermal resistance model as much as possible. In accordance with above inference, Flotherm modelling and simulation is carried out for certain printing circuit board, and computing results are compared with actual testing data to verify the correction of analysis methods.

Trends in Primary Antibiotic Resistance in H. pylori Strains Isolated in Italy between 2009 and 2019.

Background and aims: the increasing prevalence of strains resistant to antimicrobial agents is a critical issue for the management of Helicobacter pylori infection. This study aimed to evaluate, in Italian naïve patients, H. pylori antibiotic resistance trends and their potential predictive factors during the last decade. Methods: consecutive Italian naïve H. pylori positive patients, referred from General Practitioners to our Unit from January 2009 to January 2019 to perform an upper gastrointestinal endoscopy (UGIE), were considered. Each patient underwent 13C-urea breath test (13C-UBT) and UGIE with multiple biopsies to perform rapid urease test (RUT), culture/susceptibility test (vs. clarithromycin, metronidazole, levofloxacin), and histopathological examination. H. pylori status was assessed through CRM (composite reference method: at least two tests positive or only culture positive). Results: between 2009 and 2014, 1763 patients were diagnosed as H. pylori positive, 907 were naïve with antibiogram available. Between 2015 and 2019, 1415 patients were diagnosed as H. pylori positive, antibiotic susceptibility test was available in 739 naïve patients. H. pylori primary antibiotic resistance rates in the first and second five-year period were, respectively, clarithromycin 30.2% (95% CI 27.2–33.3), 37.8% (95% CI 34.2–41.4); metronidazole 33.3% (95% CI 30.2–36.5), 33.6% (95% CI 30.2–37.1); levofloxacin 25.6% (95% CI 22.8–28.5), 33.8% (95% CI 37.4–47.4), double resistance clarithromycin-metronidazole 18.9% (95% CI 16.4–21.6), 20.7% (95% CI 17.8–23.8). The increase of the resistance rates to clarithromycin and levofloxacin in naïve patients was statistically significant (p < 0.05). Although eradication rates for sequential therapy in the 10 years considered were 93.4% (95% CI 92–94.6) and 87.5% (95% CI 85.7–89) at per-protocol (PP) and intention-to-treat (ITT) analysis, respectively, they showed a significant decrease in the second five-year period. Conclusions: this data highlights an increase in primary H. pylori antibiotic resistance and strongly suggests the importance of drug susceptibility testing also in naïve patients.

Delafloxacin against Helicobacter pylori, a potential option for improving eradication success?

Increase in Helicobacter pylori resistance to fluoroquinolones has been reported in many countries. The aim of the study was to compare, for the first time to our knowledge, levofloxacin and delafloxacin activities against H. pylori, including numerous levofloxacin- and multidrug resistant strains. Minimal inhibitory concentrations (MICs) of six antibiotics against 71 consecutive clinical strains were determined. Delafloxacin MIC50 and MIC90 were 0.016 and 0.125 μg/mL versus 0.125 and ≥32 μg/mL, respectively, for levofloxacin. Against the 19 levofloxacin resistant strains, delafloxacin MICs50 and MICs90 were 0.094 and 0.38 μg/mL, respectively. Delafloxacin MICs against the 21 strains with double or multidrug resistance were ≤0.75 μg/mL. The low MICs, the activity against levofloxacin resistant and multidrug resistant H. pylori strains and the increased activity of the agent in acidic conditions make delafloxacin worthy of further investigation, aiming at optimizing fluoroquinolone-based eradication regimens.

Broad spectrum resistance in Helicobacter pylori isolated from gastric biopsies of patients with dyspepsia in Cameroon and efflux-mediated multiresistance detection in MDR isolates

BackgroundAntibiotic resistance is a leading cause of treatment failure in Helicobacter pylori infection. In Africa, there are very little data concerning the susceptibility of Helicobacter pylori isolates to antibiotics. The purpose of this study was to evaluate the resistance prevalence of Helicobacter pylori strains circulating in Cameroon, and to assess overexpression of efflux pump as a possible multi-drug resistance mechanisms.MethodsA total of 140 H. pylori isolates were recovered from gastric biopsies of dyspeptic patients in two reference hospitals in Cameroon and analyzed for their antimicrobial susceptibility to amoxicillin, co-amoxiclav, ampicillin, penicillin, imipenem, metronidazole, rifabutin, erythromycin, clarithromycin, azithromycin, levofloxacin, ciprofloxacin, norfloxacin, tetracycline, doxycycline and minocycline. Antibiotic sensitivity was tested by disk diffusion method. Phe-Arg-naphthylamide (PAßN) was used as efflux pump inhibitor. INT broth microdilution method in supplemented Brain Heart Infusion broth was used to determine the MIC of ampicillin, amoxicillin, metronidazole, erythromycin, clarithromycin and doxycycline in the absence and the presence of PAßN against 32 selected MDR isolates.ResultsOverall H. pylori resistance rate was 100% to ampicillin, penicillin and co-amoxiclav; 97.14% to amoxicillin, 97.85% to metronidazole, 47.85% to erythromycin, 13.57% to clarithromycin; 5, 2.86 and 0.71% to doxycycline, tetracycline and minocycline respectively. No resistance to azithromycin, rifabutin, imipenem, ciprofloxacin, norfloxacin and levofloxacin was detected among H. pylori isolates. Seventy percent (70%) of the tested isolates elicited a multiple drugs resistance pattern; 42.57% double, 15.71% triple and 5.71% quadruple drugs resistance. Metronidazole and amoxicillin were more concerned with double resistance pattern (86.76%). The spectrum of activity recorded with metronidazole, doxycycline, clarithromycin and erythromycin ranged from 0 to 100% in the absence to the presence of PAßN against the tested MDR isolates. An 8 to 128-fold increase in potency was also noticed with these antibiotics in the presence of PAßN.ConclusionWith regard to the high resistance rate to both amoxicillin and metronidazole, these drugs should be avoided as components of triple therapy in our milieu. In contrast, ciprofloxacin, norfloxacin, levofloxacin and tetracyclines could be used to achieve a better eradication rate and to reduce the risk of selection of H. pylori resistant strains.

Bi-allelic Homology-Directed Repair with Helper-Dependent Adenoviruses

We describe a strategy to achieve footprintless bi-allelic homology-directed repair (HDR) using helper-dependent adenoviruses (HDAds). This approach utilizes two HDAds to deliver the donor DNA. These two HDAds are identical except for their selectable marker. One expresses the puromycin N-acetyltransferase-herpes simplex virus I thymidine kinase fusion gene (PACTk), while the other expresses the hygromycin phosphotransferase-herpes simplex virus I thymidine kinase fusion gene (HyTk). Therefore, puromycin and hygromycin double resistance can be used to select for targeted HDAd integration into both alleles. Subsequently, piggyBac-mediated excision of both PACTk and HyTk will confer resistance to gancyclovir, resulting in footprintless HDR at both alleles. However, gene-targeting frequency was not high enough to achieve simultaneous targeting at both alleles. Instead, sequential targeting, whereby the two alleles were targeted one at a time, was required in order to achieve bi-allelic HDR with HDAd.