Risk Of Developing Resistance Research Articles

Acute toxicity of two insecticides on two species of Chagas disease vectors

The control of triatomine vectors of Chagas disease is mainly based on the use of pyrethroid insecticides. Because chemical control is the primary method for managing these insects, it is crucial to diversify the range of products utilized to mitigate the risk of resistance development. This study evaluated the toxicity of two insecticides with different modes of action on Triatoma dimidiata Latreille and T. pallidipennis Stal first and third instar nymphs. Our study focused on the effects of two insecticides, buprofezin (a growth regulator) and flunocamid (an anti-feeder), on the mortality rate of triatomine bugs in a laboratory setting. Moreover, we investigated how direct and indirect (film method) exposure to these insecticides impacted the survival of the insects. Flonicamid emerged as a promising insecticide for triatomine control since it caused 100% mortality in first-instar nymphs 48 h after direct exposure. While, in third instar nymphs, the maximum mortality was 88% at 72 h after exposure. Our result can be used as a basis for future triatomine control plans.

Antimicrobial blue light as a biofilm management therapy at the skin-implant interface in an ex vivo percutaneous osseointegrated implant model.

Biofilm contamination is often present at the skin-implant interface of transfemoral osseointegrated implants leading to frequent infection, irritation, and discomfort. New biofilm management regimens are needed as the current standard of washing the site with soap and water is inadequate to manage infection rates. We investigated the potential of antimicrobial blue light, which has reduced risk of resistance development and broad antimicrobial mechanisms. Our lab developed an antimicrobial blue light(aBL) device uniquely designed for an ex vivo system based on an established ovine osseointegrated (OI) implant model with Staphylococcus aureus ATCC 6538 biofilms as initial inocula. Samples were irradiated with aBL or washed for three consecutive days after which they were quantified. Colony-forming unit (CFU) counts were compared with a control group (bacterial inocula without treatment). After 1 day, aBL administered as a single 6 h dose or two 1 h doses spaced 6 h apart both reduced the CFU count by 1.63 log10 ± 0.02 CFU. Over 3 days of treatment, a positive aBL trend was observed with a maximum reduction of ~2.7 log10 CFU following 6 h of treatment, indicating a relation between multiple days of irradiation and greater CFU reductions. aBL was more effective at reducing the biofilm burden at the skin-implant interface compared with the wash group, demonstrating the potential of aBL as a biofilm management option.

Resistance to the Bacteriocin Lcn972 Deciphered by Genome Sequencing.

In view of the current threat of antibiotic resistance, new antimicrobials with low risk of resistance development are demanded. Lcn972 is a lactococcal bacteriocin that inhibits septum formation by binding to the cell wall precursor lipid II in Lactococcus. It has a species-specific spectrum of activity, making Lcn972 an attractive template to develop or improve existing antibiotics. The aim of this work was to identify mutations present in the Lcn972-resistant clone Lactococcus cremoris D1-20, previously evolved from the sensitive strain L. cremoris MG1614. Whole-genome sequencing and comparison over the reference genome L. cremoris MG1363 identified several unexpected mutations in the parental strain MG1614, likely selected during in-house propagation. In the Lcn972R clone, two previously identified mutations were mapped and confirmed. Additionally, another transposition event deregulating cellobiose uptake was identified along with three point mutations of unknown consequences for Lcn972 resistance. Two new independent evolution experiments exposing L. cremoris MG1614 to Lcn972 revealed transposition of IS981 into the LLMG_RS12285 locus as the predominant mutation selected by Lcn972. This event occurs early during evolution and was found in 100% of the evolved clones, while other mutations were not selected. Therefore, activation of LLMG_RS12285 coding for a putative anti-ECF (extra-cytoplasmic function) sigma factor is regarded as the main Lcn972 resistance factor in L. cremoris MG1614.

Low Concentrations of Eucalyptus Essential Oil Induce Age, Sex, and Mating Status-Dependent Stimulatory Responses in Drosophila suzukii

Plant-extracted essential oils are generally suggested as potential sources for alternatives to synthetic insecticides in insect pest control strategies. The increased interest in the use of essential oils derives from the generalized perception of their safety for the environment, human health, and non-target organisms as well as a lower risk of resistance development. However, studies on essential oils have largely focused on their activity on targeted insect pests while overlooking their potential unintended effects on insect biological and reproductive traits, especially with sublethal exposures. Here, we first determined the toxicity of Eucalyptus globulus essential oil to adults of Drosophila suzukii and assessed the effects of low concentrations (i.e., LC5 and LC20) in old (5–7 days) and mated flies. Subsequently, we assessed longevity and fecundity in newly emerged virgin flies from four couples’ combinations: unexposed couples, exposed females, exposed males, and exposed couples to the low concentration LC20. Our results show that eucalyptus essential oil has good insecticidal activity against adults of D. suzukii. However, compared to untreated flies, the exposure to low concentrations enhanced the females’ fecundity only when both old and mated female and male flies were exposed, while the females’ but not males’ life span was extended only in couples where newly emerged virgin females were exposed. Our findings suggest that although the eucalyptus essential oil may be a good control alternative for adult D. suzukii, its age-, sex-, and mating status-dependent stimulatory responses mediated by exposure to low concentrations need to be considered and further investigated.

L’asthme, le cardiologue, les médicaments : questions soulevées par les macrolides

Erythromycin was discovered in the early 1950s, the development in the 1980s of new generation macrolides with an improvement in their pharmacokinetic properties and their tolerance led to a renewed interest in this family of antibiotics. They are indicated in respiratory tract infections, sexually transmitted diseases and gastrointestinal infections. In addition to their antibacterial property, macrolides would have antiviral and also immunomodulatory properties, which would have motivated the search for new therapeutic indications. After the discovery of the activity of erythromycin on diffuse panbronchiolitis with reduced symptoms and improved life expectancy, studies have shown that macrolides reduce exacerbations in patients with COPD, asthma, bronchiectasis and cystic fibrosis. Macrolides have also been proposed in the treatment of coronary heart disease. In severe asthma, azithromycin has been proposed in certain endotypes as prolonged treatment for several months, the efficacy would be related to an immunomodulatory effect. Prolonged prescription of macrolides, in addition to the risk of resistance development, increases adverse effects. Apart the frequent digestive side effects, macrolides can lead to a prolongation of the QT interval. There is then a risk of torsades de pointes, ventricular tachycardia and ventricular fibrillation. The highest risk is observed with erythromycin, the lowest with azithromycin. Patients with cardiac disorders, as well as those taking anti-arrhythmics or drugs known to cause interactions, should be carefully monitored.

Fungicidal Activity of Zinc Oxide Nanoparticles against Azole-Resistant Aspergillus flavus Isolated from Yellow and White Maize.

The risk of resistance development and adverse effects on human health and the environment has increased in the last decade. Furthermore, many antifungal agents fail to inhibit the pathogenesis of azole-resistant Aspergillus flavus. In this report, we isolated and identified azole-resistant A. flavus isolates from two sources of maize (white and yellow maize). The susceptibilities of Aspergillus flavus isolates were investigated by conventional antifungals such as Terbinfine, Fluconazole, Ketoconazole, Voricazole, Amphotericin, and Nystatin. Then zinc oxide nanoparticles associated with Chlorella vulgaris, which are synthesized by using the precipitation method, were examined against isolated fungi. The results showed that twelve species of white corn were isolated out of fifty isolates, while the number of isolates from the yellow corn source was only four. Interestingly, the following antifungals have an impact effect against azole-resistant A. flavus isolates: the inhibition zones of ketoconazole, voricazole, and terbinafine were 40 mm, 20 mm, and 12 mm, respectively, while the remaining antifungal agents have no effect. Similarly, the inhibition zones of the following antifungal agents were as follows: 41 mm for Terbinfine, 13 mm for Voricazole, and 11 mm for Ketoconazole against Aspergillus flavus that was isolated from yellow corn. The physiochemical characterization of zinc oxide nanoparticles provides evidence that ZnO-NPs associate with Chlorella vulgaris and have been fabricated by the precipitation method with a diameter of 25 nm. The zinc oxide nanoparticle was then used to isolate azole-resistant A. flavus, and the results show that ZnO-NPs have an effect on azole-resistant A. flavus isolation. The inhibition zone of zinc oxide nanoparticles against A. flavus (that was isolated from white corn) was 50 mm with an MIC of 50 mg/mL, while the inhibition zone of zinc oxide nanoparticles against Azole-resistant A. flavus isolated from yellow corn was 14 nm with an MIC of 25 mg/mL, which indicated that zinc oxide nanoparticles gave a better result against Azole-resistant A. flavus isolated from maize.

Antibiotic perseverance increases the risk of resistance development

The rise of antibiotic-resistant bacterial infections poses a global threat. Antibiotic resistance development is generally studied in batch cultures which conceals the heterogeneity in cellular responses. Using single-cell imaging, we studied the growth response of Escherichia coli to sub-inhibitory and inhibitory concentrations of nine antibiotics. We found that the heterogeneity in growth increases more than what is expected from growth rate reduction for three out of the nine antibiotics tested. For two antibiotics (rifampicin and nitrofurantoin), we found that sub-populations were able to maintain growth at lethal antibiotic concentrations for up to 10 generations. This perseverance of growth increased the population size and led to an up to 40-fold increase in the frequency of antibiotic resistance mutations in gram-negative and gram-positive species. We conclude that antibiotic perseverance is a common phenomenon that has the potential to impact antibiotic resistance development across pathogenic bacteria.

Selection for resistance to pirimiphos-methyl, permethrin and spinosad in a field strain of Sitophilus oryzae: resistance risk assessment, cross-resistance potential and synergism of insecticides.

Indiscriminate use of insecticides in food storage facilities for controlling insect pests has deleterious effects on the environment and non-targeted organisms in the premises. Continuous use of insecticides may result in resistance development in insect pests, which compel the stakeholders to increase the dosage of insecticides to manage resistant insect pests. The increased dosage of insecticides ultimately may result in contamination of stored food stuff that affects human health. The present study was planned to generate data that will be helpful to delay resistance development and to reduce environmental pollution. A field strain of Sitophilus oryzae, one of the most common insect pests of stored foodstuff, was selected separately with pirimiphos-methyl, permethrin, or spinosad for five consecutive generations. The selected strains were studied for resistance risk assessment, time taken to develop resistance to insecticides after continuous exposure in the selection process, preliminary mechanism of resistance, and whether the development of resistance due to the selection with a particular insecticide could develop cross-resistance to other insecticide or not. In comparison to a laboratory susceptible reference strain, the insecticide-selected strains revealed rapid development of resistance against insecticides as a result of selection process: 31.05-fold resistance to pirimiphos-methyl, 156.49-fold resistance to permethrin, and 65.6-fold resistance to spinosad. The selected strains did not show cross-resistance to insecticides to with these strains were not exposed during selection experiments, i.e., strain selected with pirimiphos-methyl did not show cross-resistance to spinosad and permethrin. In the synergism bioassays, the synergists (S,S,S-tributyl phosphorotrithioate and piperonyl butoxide) significantly reduced resistance of the selected strain against insecticides to with these were selected, revealing the probability of metabolic mechanism of resistance. The present study revealed high risks of resistance development to pirimiphos-methyl, spinosad, and permethrin under consistent selection pressure. Lack of cross-resistance among insecticides provides an opportunity to use insecticides in rotation instead of increasing dosages to manage resistant insects that will ultimately pollute the environment.

Insights into antibiotic stewardship of lake-rivers-basin complex systems for resistance risk control

Antibiotic stewardship is hindered by a lack of consideration for complicated environmental fate of antibiotics and their role in resistance development, while the current methodology of eco-toxicological risk assessment has not been fully protective against their potential to select for antibiotic resistance. To address this problem, we established a novel methodologic framework to perform comprehensive environmental risk assessment of antibiotics in terms of resistance development, which was based on selection effect, phenotype resistance level, heteroresistance frequency, as well as prevalence and stability of antibiotic resistance genes. We tracked the contribution of antibiotic load reduction to the mitigation of environmental risk of resistance development by fate and transport modeling. The method was instantiated in a lake-river network-basin complex system, taking the Taihu Basin as a case study. Overall, antibiotic load posed no eco-toxicological risk but an average medium-level environmental risk for resistance development in Taihu Lake. The effect of antibiotic load on resistance risk was both seasonal-dependent and category-dependent, while quinolones posed the greatest environmental risk for resistance development. Mass-flow analysis indicated that temporal-spatial variation in hydrological regime and antibiotic fate together exerted a significant effect on antibiotic load in the system. By apportioning antibiotic load to riverine influx, we identified the hotspots for load reduction and predicted the beneficial response of resistance risk under load-reduction scenarios. Our study proposed a risk-oriented strategy of basin-scaled antibiotic load reduction for environmental risk control of resistance development.

Risk ranking of macrolide antibiotics – Release levels, resistance formation potential and ecological risk

Antibiotic resistance (AR) development in natural water bodies is a significant source of concern. Macrolide antibiotics in particular have been identified as pollutants of concern for AR development throughout the literature, as well as by state and international authorities. This study utilises a probabilistic model to examine the risk of AR development arising from human-use macrolide residues, utilising administration rates from Ireland as a case study. Stages modelled included level of administration, excretion, degradation in wastewater, removal in wastewater treatment, assuming conventional activated sludge (CAS) treatment, and dilution. Release estimates per day, as well as risk quotient values for antibiotic resistance development and ecological impact, are generated for erythromycin, clarithromycin, and azithromycin. In the modelled scenario in which conventional activated sludge treatment is utilised in wastewater treatment, this model ranks risk of resistance development for each antibiotic in the order clarithromycin > azithromycin > erythromycin, with mean risk quotient values of 0.50, 0.34 and 0.12, respectively. A membrane bioreactor scenario was also modelled, which reduced risk quotient values for all three macrolides by at least 50 %. Risk of ecological impact for each antibiotic was also examined, by comparing environmental concentrations predicted to safety limits based on toxicity data for cyanobacteria and other organisms from the literature, with azithromycin being identified as the macrolide of highest risk. This study compares and quantifies the risk of resistance development and ecological impact for a high-risk antibiotic group in the Irish context, and demonstrates the potential for risk reduction achieved by adoption of alternative (e.g. membrane bioreactor) technology.

Synergy of cystamine and pyraclostrobin against Fusarium graminearum involves membrane permeability mitigation and autophagy enhancement

The application of fungicide mixture is one of the most important measures to extend the service life of highly selective fungicides. Pyraclostrobin (PYR), which has been extensively used to control plant diseases by inhibiting mitochondrial respiration of pathogenic fungi, is at a high risk of resistance development. In this study, the potential of PYR alone or in combination with cystamine, an inhibitor of microbial transglutaminase, to suppress Fusarium graminearum was tested in vitro and in vivo. A synergistic effect of PYR/CYS mixture was observed both in vitro and when applied to etiolated wheat coleoptile. The control effect of PYR/CYS mixture on F. graminearum was better than that of PYR alone, which was reflected by the increased protection effect. The discrepancies of membrane permeability and the redox-physiological state were observed between PYR and PYR/CYS treatments, suggesting that an increased PYR availability in F. graminearum mycelia could be related with the observed synergistic action. Moreover, a synergistic profile was observed between PYR and CYS in regard of massive autophagosomes in mycelia, indicating that enhanced autophagy could be involved in the mode of action of PYR/CYS mixture. The differential content of mitochondrial metabolites between PYR and PYR/CYS treatments also provided evidence for CYS contribution to the fungicidal action of PYR/CYS mixture. The results provide insight into the synergistic mechanism of action of PYR/CYS mixture and an effective way to enhance the efficiency of PYR to combat F. graminearum.

Integrated Use of Herbicides and Mulching for Sustainable Control of Purple Nutsedge (Cyperus rotundus) in a Tomato Crop

Purple nutsedge (Cyperus rotundus L.) is a problematic weed in tomato (Lycopersicon esculentum L.) crops causing significant yield losses. Although several chemical options are available for this weed, the level of control is often unsatisfactory, and the consistent use of herbicides has a risk of resistance evolution. Therefore, we evaluated the efficacy of two herbicide options, halosulfuron and fomesafen + S-metolachlor, alone or integrated with natural and plastic mulches in controlling purple nutsedge in tomato crops in a three-year field study. The use of herbicides or mulches alone did not provide effective weed control (below 65%). However, the combination of natural mulch and fomesafen + S-metolachlor provided the most effective weed control by reducing the density and biomass of purple nutsedge by up to 83% and 81%, respectively, as compared with the season-long untreated control. The use of a natural mulch in combination with the herbicides halosulfuron or fomesafen + S-metolachlor also resulted in the highest tomato yield (ca. 3.3 kg per plant). This integrated treatment improved tomato yield by over 400% as compared with the season-long untreated control. The integrated use of a plastic mulch and two chemical options resulted in a 67–74% weed biomass reduction and a 332–368% yield increase over the season-long untreated control. These findings suggest that the combinations of herbicides and mulches are effective integrated weed management options for purple nutsedge in tomato crops.

New In Vitro Interaction-Parasite Reduction Ratio Assay for Early Derisk in Clinical Development of Antimalarial Combinations.

The development and spread of drug-resistant phenotypes substantially threaten malaria control efforts. Combination therapies have the potential to minimize the risk of resistance development but require intensive preclinical studies to determine optimal combination and dosing regimens. To support the selection of new combinations, we developed a novel in vitro-in silico combination approach to help identify the pharmacodynamic interactions of the two antimalarial drugs in a combination which can be plugged into a pharmacokinetic/pharmacodynamic model built with human monotherapy parasitological data to predict the parasitological endpoints of the combination. This makes it possible to optimally select drug combinations and doses for the clinical development of antimalarials. With this assay, we successfully predicted the endpoints of two phase 2 clinical trials in patients with the artefenomel-piperaquine and artefenomel-ferroquine drug combinations. In addition, the predictive performance of our novel in vitro model was equivalent to that of the humanized mouse model outcome. Last, our more informative in vitro combination assay provided additional insights into the pharmacodynamic drug interactions compared to the in vivo systems, e.g., a concentration-dependent change in the maximum killing effect (Emax) and the concentration producing 50% of the killing maximum effect (EC50) of piperaquine or artefenomel or a directional reduction of the EC50 of ferroquine by artefenomel and a directional reduction of Emax of ferroquine by artefenomel. Overall, this novel in vitro-in silico-based technology will significantly improve and streamline the economic development of new drug combinations for malaria and potentially also in other therapeutic areas.

Resistance risk assessment in diamondback moth, Plutella xylostella (L.) to fluxametamide

The diamondback moth Plutella xylostella (L.) (Lepidoptera: Plutellidae) is a devastating pest that infests several cruciferous vegetable crops worldwide and has developed insecticide resistance in most chemical classes. Fluxametamide is a novel neuroactive isoxazoline compound that binds to a unique site on the γ-aminobutyric acid (GABA) receptor and has an exceptional insecticidal activity on a range of lepidopteran caterpillars. The present study aimed to assess the risk of resistance in P. xylostella to fluxametamide under laboratory conditions. After the repeated exposure to fluxametamide for successive 18 generations of P. xylostella, there was 3.095-fold increase in the LC50. When the realized heritability (h2) of P. xylostella to fluxametamide in the open field was assumed to be the laboratory-estimated value (h2 = 0.180) and the mortality was 70–90%, only 22.9–33.1 generations were expected to be required to obtain a 10-fold increase in fluxametamide resistance. Compared with the laboratory susceptible strain (F0), the Flux-SEL (F18) strain exhibited a very low level of cross-resistance to emamectin benzoate (5.17-fold) and no significant cross-resistance to fipronil or flubendiamide. With the F0 and the Flux-SEL test strains (F12, F15 and F18), the specific activities of cytochrome P450 and glutathione S-transferase showed a remarkable increase during selection, while the esterase activity did not differ significantly. The results indicated that a potential risk of resistance development to fluxametamide exists in P. xylostella after continuous exposure. These pieces of information will be crucial in formulating rational fluxametamide application and resistance management guidelines for controlling P. xylostella under field condition.

A tiered probabilistic approach to assess antibiotic ecological and resistance development risks in the fresh surface waters of China

Exposure to antibiotics can result in not only ecotoxicity on aquatic organisms but also the development of antibiotic resistance. In the study, the ecotoxicity data and minimum inhibitory concentrations of the antibiotics were screened to derive predicted no-effect concentrations of ecological (PNECeco) and resistance development risks (PNECres) for 36 antibiotics in fresh surface waters of China. The derived PNECeco and PNECres values were ranged from 0.00175 to 2351 μg/L and 0.037–50 μg/L, respectively. Antibiotic ecological and resistance development risks were geographically widespread, especially in the Yongding River, Daqing River, and Ziya River basins of China. Based on the risk quotients, 11 and 14 of 36 target antibiotics were at high ecological risks and high resistance development risks in at least one basin, respectively. The higher tiered assessments provided more detailed risk descriptions by probability values and β-lactams (penicillin and amoxicillin) were present at the highest levels for ecological and resistance development risks. Although there was uncertainty based on the limited data and existing methods, this study can indicate the overall situation of the existing risk levels and provide essential insights and data supporting antibiotic management.

The Cross-Resistance Pattern and the Metabolic Resistance Mechanism of Acetamiprid in the Brown Planthopper, Nilaparvata lugens (Stål).

Acetamiprid is widely used in paddy fields for controlling Nilaparvata lugens (Stål). However, the risk of resistance development, the cross-resistance pattern and the resistance mechanism of acetamiprid in this pest remain unclear. In this study, an acetamiprid-resistant strain (AC-R) was originated from a field strain (UNSEL) through successive selection with acetamiprid for 30 generations, which reached 60.0-fold resistance when compared with a laboratory susceptible strain (AC-S). The AC-R strain (G30) exhibited cross-resistance to thiamethoxam (25.6-fold), nitenpyram (21.4-fold), imidacloprid (14.6-fold), cycloxaprid (11.8-fold), dinotefuran (8.7-fold), sulfoxaflor (7.6-fold) and isoprocarb (8.22-fold), while there was no cross-resistance to etofenprox, buprofezin and chlorpyrifos. Acetamiprid was synergized by the inhibitor piperonyl butoxide (2.2-fold) and the activity of cytochrome P450 monooxygenase was significantly higher in the AC-R strain compared with the AC-S strain, suggesting the critical role of P450. The gene expression results showed that the P450 gene CYP6ER1 was significantly overexpressed in AC-R compared with the AC-S and UNSEL strains. In addition, the RNA interference (RNAi) of CYP6ER1 significantly increased the susceptibility of AC-R to acetamiprid. Molecular docking predicted that acetamiprid and CYP6ER1 had close binding sites, and the nitrogen atoms had hydrogen bond interactions with CYP6ER1. These results demonstrated that the overexpression of CYP6ER1 contributed to acetamiprid resistance in N. lugens.

Mode of action of elasnin as biofilm formation eradicator of methicillin-resistant Staphylococcus aureus.

Biofilm is made up of microbes and their extracellular matrix, making microorganisms highly tolerant, resistant, and resilient to a wide range of antimicrobials. Biofilm treatment with conventional antimicrobial agents can accelerate the evolution and spread of resistance due to the reduced efficacy and increased gene transfer and differentiation within biofilms. Therefore, effective biofilm-targeting compounds are currently highly sought after. In the present study, we identified elasnin as a potent biofilm-targeting compound against methicillin-resistant Staphylococcus aureus (MRSA). Elasnin effectively inhibited biofilm formation and especially eradicated the pre-formed biofilms of MRSA with low cytotoxicity and low risk of resistance development and retains its activity in a chronic wound biofilms model. A comprehensive mechanistic study using multi-omics and confocal and scanning electron microscopy revealed that elasnin induced the biofilm matrix destruction in a time-dependent manner and interfered with the cell division during the exponential phase, primarily by repressing the expression of virulence factors. Cells released from the elasnin-treated biofilms exhibited a defective appearance and became more sensitive to beta-lactam antibiotic penicillin G. Through gene overexpression and deletion assay, we discovered the key role of sarZ during elasnin-induced biofilm eradication. Overall, the present study identified elasnin as a potent biofilm eradicator against MRSA that harbors potential to be developed for biofilm removal and chronic wound treatment, and provided new insights into the molecular targets for biofilm eradication in MRSA.

In silico genome-scale metabolic modeling and in vitro static time-kill studies of exogenous metabolites alone and with polymyxin B against Klebsiella pneumoniae.

Multidrug-resistant (MDR) Klebsiella pneumoniae is a top-prioritized Gram-negative pathogen with a high incidence in hospital-acquired infections. Polymyxins have resurged as a last-line therapy to combat Gram-negative “superbugs”, including MDR K. pneumoniae. However, the emergence of polymyxin resistance has increasingly been reported over the past decades when used as monotherapy, and thus combination therapy with non-antibiotics (e.g., metabolites) becomes a promising approach owing to the lower risk of resistance development. Genome-scale metabolic models (GSMMs) were constructed to delineate the altered metabolism of New Delhi metallo-β-lactamase- or extended spectrum β-lactamase-producing K. pneumoniae strains upon addition of exogenous metabolites in media. The metabolites that caused significant metabolic perturbations were then selected to examine their adjuvant effects using in vitro static time–kill studies. Metabolic network simulation shows that feeding of 3-phosphoglycerate and ribose 5-phosphate would lead to enhanced central carbon metabolism, ATP demand, and energy consumption, which is converged with metabolic disruptions by polymyxin treatment. Further static time–kill studies demonstrated enhanced antimicrobial killing of 10 mM 3-phosphoglycerate (1.26 and 1.82 log10 CFU/ml) and 10 mM ribose 5-phosphate (0.53 and 0.91 log10 CFU/ml) combination with 2 mg/L polymyxin B against K. pneumoniae strains. Overall, exogenous metabolite feeding could possibly improve polymyxin B activity via metabolic modulation and hence offers an attractive approach to enhance polymyxin B efficacy. With the application of GSMM in bridging the metabolic analysis and time–kill assay, biological insights into metabolite feeding can be inferred from comparative analyses of both results. Taken together, a systematic framework has been developed to facilitate the clinical translation of antibiotic-resistant infection management.

Phenotypic and genotypic characterisation of thymine auxotrophy in Escherichia coli isolated from a patient with recurrent bloodstream infection.

IntroductionThymine auxotrophic in vitro mutants of Escherichia coli were first reported in the mid-20th century. Later, thymine-dependent clinical strains of E. coli as well as other Enterobacterales, Enterococcus faecalis and Staphylococcus aureus have been recognized as the cause of persistent and recurrent infections.ObjectivesThe aim of this study was to characterize the phenotype and investigate the molecular basis of thymine auxotrophy in ten E. coli isolates obtained at different time points from a patient with recurrent bloodstream infection (BSI) due to a chronic aortic graft infection treated with Trimethoprim/sulfamethoxazole (TMP-SMX).MethodsClinical data was obtained from hospital records. Growth characterization and antimicrobial susceptibility testing to TMP-SMX was performed on M9 agar and in MH broth with different thymine concentrations (0.5, 2, 5, 10 and 20 μg/mL), on Mueller-Hinton (MH) and blood agar. Whole genome sequencing (WGS) was performed on all E. coli isolates.ResultsE. coli were isolated from ten consecutive BSI episodes from a patient with chronic aortic graft infection. Six of these isolates were resistant to TMP-SMX when assayed on blood agar. Growth experiments with added thymine confirmed that these isolates were thymine-dependent (thy-), and revealed growth defects (slower growth rate and smaller colony size) in these isolates relative to thy+ isolates (n = 4). WGS indicated that all isolates were of the same clonal lineage of sequence type 7358. Genomic analysis revealed a G172C substitution in thyA in all TMP-SMX resistant isolates, while mutations affecting genes involved in the deoxyribose salvage pathway (deoB and deoC) were identified in eight isolates.ConclusionThis case highlights the risk of resistance development to TMP-SMX, especially for long-term treatment, and the possible pitfalls in detection of growth-deficient subpopulations from chronic infections, which could lead to treatment failure.

Rapid Detection of Bacterial and Fungal Pathogens Using the T2MR versus Blood Culture in Patients with Severe COVID-19.

ABSTRACTA high rate of bacterial and fungal superinfections was reported in critically ill patients with COVID-19. However, diagnosis can be challenging. The aim of this study is to evaluate the sensitivity and the clinical utility of the point-of-care method T2 magnetic resonance (T2MR) with the gold standard: the blood culture. T2MR can potentially detect five different Candida species and six common bacteria (so-called “ESKAPE” pathogens including Escherichia coli, Staphylococcus aureus, Klebsiella pneumoniae, Acinet`obacter baumanii, Pseudomonas aeruginosa, and Enterococcus faecium). If superinfection was suspected in patients with COVID-19 admitted to the intensive care unit, blood culture and two panels of T2MR were performed. Eighty-five diagnostic bundles were performed in 60 patients in total. T2MR detected an ESKAPE pathogen in 9 out of 85 (10.6%) samples, compared to BC in 3 out of 85 (3.5%). A Candida species was detected in 7 of 85 (8.2%) samples of T2MR compared to 1 out of 85(1.2%) in blood culture. The mean time to positive test result in samples with concordant positive results was 4.5 h with T2MR and 52.5 h with blood culture. The additional use of T2MR enables a highly sensitive and rapid detection of ESKAPE and Candida pathogens.IMPORTANCE Coronavirus disease 2019 (COVID-19) has led to a high number of deaths since the beginning of the pandemic worldwide. One of the reasons is the high number of bacterial and fungal superinfections in patients suffering from critical disease. However, diagnosis is often challenging. In this study we could show that the additional use of the culture-independent method T2MR did not only show a much higher detection rate of bacterial and fungal pathogens but also a significantly shorter time until detection and therapy change compared to the gold standard: the blood culture. The implementation of T2MRin the care of patients with severe course of COVID-19 might lead to an earlier sufficient antimicrobial therapy and as a result lower mortality and less use of broad-spectrum unnecessary therapy reducing the risk of resistance development.