Uptake Of Antibiotics Research Articles

Breaching the Barrier: Genome-Wide Investigation into the Role of a Primary Amine in Promoting E. coli Outer-Membrane Passage and Growth Inhibition by Ampicillin.

Gram-negative bacteria are problematic for antibiotic development due to the low permeability of their cell envelopes. To rationally design new antibiotics capable of breaching this barrier, more information is required about the specific components of the cell envelope that prevent the passage of compounds with different physiochemical properties. Ampicillin and benzylpenicillin are β-lactam antibiotics with identical chemical structures except for a clever synthetic addition of a primary amine group in ampicillin, which promotes its accumulation in Gram-negatives. Previous work showed that ampicillin is better able to pass through the outer membrane porin OmpF in Escherichia coli compared to benzylpenicillin. It is not known, however, how the primary amine may affect interaction with other cell envelope components. This study applied TraDIS to identify genes that affect E. coli fitness in the presence of equivalent subinhibitory concentrations of ampicillin and benzylpenicillin, with a focus on the cell envelope. Insertions that compromised the outer membrane, particularly the lipopolysaccharide layer, were found to decrease fitness under benzylpenicillin exposure, but had less effect on fitness under ampicillin treatment. These results align with expectations if benzylpenicillin is poorly able to pass through porins. Disruption of genes encoding the AcrAB-TolC efflux system were detrimental to survival under both antibiotics, but particularly ampicillin. Indeed, insertions in these genes and regulators of acrAB-tolC expression were differentially selected under ampicillin treatment to a greater extent than insertions in ompF. These results suggest that maintaining ampicillin efflux may be more significant to E. coli survival than full inhibition of OmpF-mediated uptake. IMPORTANCE Due to the growing antibiotic resistance crisis, there is a critical need to develop new antibiotics, particularly compounds capable of targeting high-priority antibiotic-resistant Gram-negative pathogens. In order to develop new compounds capable of overcoming resistance a greater understanding of how Gram-negative bacteria are able to prevent the uptake and accumulation of many antibiotics is required. This study used a novel genome wide approach to investigate the significance of a primary amine group as a chemical feature that promotes the uptake and accumulation of compounds in the Gram-negative model organism Escherichia coli. The results support previous biochemical observations that the primary amine promotes passage through the outer membrane porin OmpF, but also highlight active efflux as a major resistance factor.

In situ precipitated hydroxyapatite-chitosan composite loaded with ciprofloxacin: Formulation, mechanical, in vitro antibiotic uptake, release, and antibacterial properties

Three-dimensional hydroxyapatite-chitosan (HA-CS) composites loaded with ciprofloxacin antibiotic (HA–CS–CIP) were formulated using the in situ and the solid-liquid method coupled with the freeze-drying process. The interaction of the HA-CS composite powder and ciprofloxacin antibiotic (CIP) was investigated by batch adsorption essays. The kinetic and the isotherm data were fitted well to the pseudo-second-order and Freundlich models, respectively. The compressive strength of the HA-CS composite was increased by 7-fold and 10-fold when using respectively 15 wt% and 30 wt% of the polymer compared to the HA-CS5 formulation (3.6 ± 0.7 MPa) made only from HA powder and CS gel (5 wt%). However, this parameter decreased from 36.8 ± 8.5 MPa down to 20.5 ± 4.7 MPa when the antibiotic content increased from 0 up to 9 wt%, respectively. The in vitro release results showed a sustained and controlled CIP release for up to 10 days. The release data fitting and modeling indicate that the process follows a Fickian diffusion mechanism. Also, the formulated composite revealed an antibacterial effect against Staphylococcus aureus and Escherichia coli bacteria. The developed composite may be a promising candidate for bone substitution and as an antibiotic local delivery system for the treatment of orthopedic implant-associated infections.

Dissemination of antibiotics through the wastewater–soil–plant–earthworm continuum

Under the ongoing climate change scenario, treated municipal wastewater (TMW) is a potential candidate for irrigated agriculture but may result in the exposure of agricultural environments to antibiotics. We studied the transfers of trimethoprim, sulfamethoxazole, and sulfapyridine in the TMW–soil–plant–earthworm continuum under greenhouse/laboratory conditions. Irrigation of potted spinach and radish with as-collected TMW resulted in no transfers of antibiotics into soil or plants owing to their low concentrations in the tertiary-treated TMW. However, TMW spiked with higher antibiotic concentrations led to transfers through this continuum. High initial inputs, slow soil degradation, and chemical speciation of the antibiotics, coupled with an extensive plant-root distribution, were important factors enhancing the plant uptake of antibiotics. In microcosm studies, transfers from vegetable materials into earthworms were low but showed potential for bioaccumulation. Such food chain transfers of antibiotics may be a driver for antibiotic resistance in agricultural systems, which is an area worthy of future study. These issues can perhaps be mitigated through high levels of TMW purification to effectively remove antibiotic compounds.

Study of pH‐dependent fluorescent property of fluoroquinolone antibiotics

AbstractAntibiotics are probably known as “wonder drugs” that inhibit bacterial growth or kill bacteria. Fluoroquinolones (FQNs) are broad‐spectrum synthetic antibiotics having excellent oral absorption and outstanding biocompatibility. It was found that some of the antibiotics themselves have a fluorescent property, and this fluorescent ability of these antibiotics has potential applications in infection diagnosis, detection of antibiotics in the environment, tracking the antibiotic uptake, and the detection of bacterial infections. In this study, we have studied the influence of pH (2–12) on fluorescence intensity, UV‐Vis absorption, and surface charge properties of fluorescent antibiotics, such as moxifloxacin, ciprofloxacin, ofloxacin, levofloxacin, and norfloxacin. The purposed study is an easy, simple way of finding the presence of antibiotics and tracking bacterial infection by changing the pH value of the medium.

Molecular Basis of Non-β-Lactam Antibiotics Resistance in Staphylococcus aureus.

Methicillin-resistant Staphylococcus aureus (MRSA) is one of the most successful human pathogens with the potential to cause significant morbidity and mortality. MRSA has acquired resistance to almost all β-lactam antibiotics, including the new-generation cephalosporins, and is often also resistant to multiple other antibiotic classes. The expression of penicillin-binding protein 2a (PBP2a) is the primary basis for β-lactams resistance by MRSA, but it is coupled with other resistance mechanisms, conferring resistance to non-β-lactam antibiotics. The multiplicity of resistance mechanisms includes target modification, enzymatic drug inactivation, and decreased antibiotic uptake or efflux. This review highlights the molecular basis of resistance to non-β-lactam antibiotics recommended to treat MRSA infections such as macrolides, lincosamides, aminoglycosides, glycopeptides, oxazolidinones, lipopeptides, and others. A thorough understanding of the molecular and biochemical basis of antibiotic resistance in clinical isolates could help in developing promising therapies and molecular detection methods of antibiotic resistance.

Loss of outer membrane protein A (OmpA) impairs the survival of Salmonella Typhimurium by inducing membrane damage in the presence of ceftazidime and meropenem.

Salmonella enterica serovar Typhimurium is one of the significant non-typhoidal Salmonella serovars that causes gastroenteritis. The rapid development of antimicrobial resistance necessitates studying new antimicrobials and their therapeutic targets in this pathogen. Our study aimed to investigate the role of four prominent outer membrane porins of S. Typhimurium, namely OmpA, OmpC, OmpD and OmpF, in developing resistance against ceftazidime and meropenem. The antibiotic-mediated inhibition of bacterial growth was determined by measuring the absorbance and the resazurin assay. DiBAC4 (Bis-(1,3-Dibutylbarbituric Acid)Trimethine Oxonol), 2,7-dichlorodihydrofluoroscein diacetate (DCFDA) and propidium iodide were used to determine the outer membrane depolarization, reactive oxygen species (ROS) generation and subsequent killing of Salmonella. The expression of oxidative stress-response and efflux pump genes was quantified by quantitative RT-qPCR. HPLC was done to determine the amount of antibiotics that entered the bacteria. The damage to the bacterial outer membrane was studied by confocal and atomic force microscopy. The in vivo efficacy of ceftazidime and meropenem were tested in the C57BL/6 mouse model. Deleting ompA reduced the survival of Salmonella in the presence of ceftazidime and meropenem. Massive outer membrane depolarization and reduced expression of oxidative stress-response genes in S. Typhimurium ΔompA hampered its growth in the presence of antibiotics. The enhanced uptake of antibiotics and decreased expression of efflux pump genes in S. Typhimurium ΔompA resulted in damage to the bacterial outer membrane. The clearance of the S. Typhimurium ΔompA from C57BL/6 mice with ceftazidime treatment proved the role of OmpA in rendering protection against β-lactam antibiotics. OmpA protects S. Typhimurium from two broad-spectrum β-lactam antibiotics, ceftazidime and meropenem, by maintaining the stability of the outer membrane.

Electrochemical Quantification of Tobramycin Retention in Pseudomonas aeruginosa as Antimicrobial Susceptibility Indicator.

The emergence and spread of bacterial resistance to antibiotics has developed into one of the most challenging threats to public health. Antibiotic susceptibility tests (ASTs) for bacterial infections are now essential, because they provide guidance for physicians in the selection of antibiotics, to which bacteria will respond. Most current AST methods require long periods of time, because of bacterial growth and incubation, leading to a prolonged and overuse of broad-spectrum antibiotics. Thus, there is a growing demand for methods and technologies that enable rapid antibiotic susceptibility assessment. Due to advantages related to cost-effectiveness, rapid response time and high sensitivity, electrochemical detection methods are promising analytical tools that can successfully quantify antibiotic uptake and retention in clinically relevant bacterial strains. This study presents the electroanalytical quantification of tobramycin (TOB) retention in susceptible and resistant bacterial strains of Pseudomonas aeruginosa. The electrochemical behavior of TOB was characterized by voltammetry, identifying redox potentials, the current dependence on pH conditions, and the detection limit at unmodified glassy carbon electrodes. The presented methodology was able to distinguish between susceptible and resistant bacterial strains, and is also capable of identifying varying degrees of resistance against TOB. The presented approach detects the immediate interaction of bacteria with an antibiotic, without the need of complex and cost-intense equipment related to genomic testing methods.

Modulatory Contribution of Oxygenating Hydrogels and Polyhexamethylene Biguanide on the Antimicrobial Potency of Neutrophil-like Cells.

Neutrophils are a first line of host defense against infection and utilize a series of oxygen-dependent processes to eliminate pathogens. Research suggests that oxygen availability can improve anti-infective mechanisms by promoting the formation of reactive oxygen species. Also, oxygen can synergistically upregulate the antibacterial properties of certain antibiotics against bacteria by altering their metabolism and causing an increase in the antibiotic uptake of bacteria. Therefore, understanding the effects of oxygen availability, as provided via a biomaterial treatment alone or along with potent antibacterial agents, on neutrophil functions can lead us to the development of new anti-inflammatory and anti-infective approaches. However, the study of neutrophil functions in vitro is often limited by their short life span and nonreproducibility, which suggests the need for cell line-based models as a substitute for primary neutrophils. Here, we took advantage of the differentiated human leukemia-60 cell line (HL-60), as an in vitro neutrophil model, to test the effects of local oxygen and antibacterial delivery by fluorinated methacrylamide chitosan (MACF) hydrogels incorporated with polyhexamethylene biguanide (PHMB) antibacterial agent. Considering the natural modes of neutrophil actions to combat bacteria, we studied the impact of our dual functioning oxygenating-antibacterial platforms on neutrophil phagocytosis and antibacterial properties as well as the formation of neutrophil extracellular traps (NETs) and reactive oxygen species (ROS). Our results demonstrated that supplemental oxygen and antibacterial delivery from MACF-PHMB hydrogel platforms upregulated neutrophil antibacterial properties and ROS production. NET formation by neutrophils upon treatment with MACF and PHMB varied when chemical and biological stimuli were used. Overall, this study presents a model to study immune responses in vitro and lays the foundation for future studies to investigate if similar responses also occur in vivo.

Patterns and trends of antibacterial treatment in patients with urinary tract infections, 2015–2019: an analysis of health insurance data

BackgroundUrinary tract infections are among the most common reason for encounter and subsequent antibiotic prescriptions. Due to the risk of collateral damage and increasing resistance rates, explicit recommendations against the use of fluoroquinolones like ciprofloxacin in uncomplicated urinary tract infections have been issued. However, to what extent these recommendations were followed and if there are relevant differences between the disciplines involved (general practitioners, urologists, paediatricians and gynaecologists) are unknown.MethodsWe used anonymized data from a local statutory health insurance (SHI) company, which covered about 38% of all SHI-insured persons in the federal state of Bremen, Germany between 2015—2019. Data included demographics, outpatient diagnoses and filled prescriptions on an individual level.ResultsOne-year prevalence of urinary tract infections was 5.8% in 2015 (females: 9.2%, males: 2.5%). Of all 102,715 UTI cases, 78.6% referred to females and 21.4% to males, 6.0% of cases were younger than 18 years. In females, general practitioners were the most common diagnosing speciality (52.2%), followed by urologists (20.0%) and gynaecologists (16.1%). Overall, fluoroquinolones were most often prescribed (26.3%), followed by fosfomycin (16.1%) and the combination of sulfamethoxazole and trimethoprim (14.2%). Fluoroquinolones were most often prescribed by urologists and general practitioners, while gynaecologists preferred fosfomycin. During the study period, shares of fluoroquinolones decreased from 29.4% to 8.7% in females and from 45.9% to 22.3% in males.ConclusionsDespite a clear trend toward a more guideline adherent prescription pattern, there is still room for improvement regarding the use of second-line antibiotics especially fluoroquinolones. The choice of antibiotics prescribed differs between specialities with higher uptake of guideline-recommended antibiotics by gynaecologists, mainly because of higher prescription shares of fosfomycin.

A Target Engagement Assay to Assess Uptake, Potency, and Retention of Antibiotics in Living Bacteria.

New antibiotics are urgently needed to counter the emergence of antimicrobial-resistant pathogenic bacteria. A major challenge in antibiotic drug discovery is to turn potent biochemical inhibitors of essential bacterial components into effective antimicrobials. This difficulty is underpinned by a lack of methods to investigate the physicochemical properties needed for candidate antibiotics to permeate the bacterial cell envelope and avoid clearance by the action of bacterial efflux pumps. To address these issues, here we used a target engagement assay to measure the equilibrium and kinetic binding parameters of antibiotics targeting dihydrofolate reductase (DHFR) in live bacteria. We also used this assay to identify novel DHFR ligands having antimicrobial activity. We validated this approach using the Gram-negative bacteria Escherichia coli and the emerging human pathogen Mycobacterium abscessus. We expect the use of target engagement assays in bacteria to expedite the discovery and progression of novel, cell-permeable antibiotics with on-target activity.

New antimicrobial peptide-antibiotic combination strategy for Pseudomonas aeruginosa inactivation.

Novel antimicrobials or new treatment strategies are urgently needed to treat Pseudomonas aeruginosa (P. aeruginosa) related infections and especially to address the problem of antibiotic resistance. We propose a novel strategy that combines the human antimicrobial peptide (AMP) LL37 with different antibiotics to find synergistic AMP-antibiotic combinations against P. aeruginosa strains in vitro. Our results showed that LL37 exhibited synergistic inhibitory and bactericidal effects against P. aeruginosa strains PAO1 and PA103 when combined with the antibiotics vancomycin, azithromycin, polymyxin B, and colistin. In addition, LL37 caused strong outer membrane permeabilization, as demonstrated through measurement of an increased uptake of the fluorescent probe N-phenyl-1-naphthylamine. The membrane permeabilization effects appear to explain why it was easier to rescue the effectiveness of the antibiotic toward the bacteria because the outer membrane of P. aeruginosa exhibits barrier function for antibiotics. Furthermore, the change in the zeta potential was measured for P. aeruginosa strains with the addition of LL37. Zeta potentials for P. aeruginosa strains PAO1 and PA103 were -40.9 and -10.9 mV, respectively. With the addition of LL37, negative zeta potentials were gradually neutralized. We found that positively charged LL37 can interact with and neutralize the negatively charged bacterial outer membrane through electrostatic interactions, and the process of neutralization is believed to have contributed to the increase in outer membrane permeability. Finally, to further illustrate the relationship between outer membrane permeabilization and the uptake of antibiotics, we used LL37 to make the outer membrane of P. aeruginosa strains more permeable, and minimum inhibitory concentrations (MICs) for several antibiotics (colistin, gentamicin, polymyxin B, vancomycin, and azithromycin) were measured. The MICs decreased were twofold to fourfold, in general. For example, the MICs of azithromycin and vancomycin decreased more than fourfold when against P. aeruginosa strain PAO1, which were the greatest decrease of any of the antibiotics tested in this experiment. As for PA103, the MIC of polymyxin B2 decreased fourfold, which was the strongest decrease seen for any of the antibiotics tested in this experiment. The increased uptake of antibiotics not only demonstrates the barrier role of the outer membrane but also validates the mechanism of synergistic effects that we have proposed. These results indicate the great potential of an LL37-antibiotic combination strategy and provide possible explanations for the mechanisms behind this synergy.

Effects of LPS Composition in Escherichia coli on Antibacterial Activity and Bacterial Uptake of Antisense Peptide-PNA Conjugates.

The physical and chemical properties of the outer membrane of Gram-negative bacteria including Escherichia coli have a significant impact on the antibacterial activity and uptake of antibiotics, including antimicrobial peptides and antisense peptide-peptide nucleic acid (PNA) conjugates. Using a defined subset of E. coli lipopolysaccharide (LPS) and envelope mutants, components of the LPS-core, which provide differential susceptibility toward a panel of bacterial penetrating peptide (BPP)-PNA conjugates, were identified. Deleting the outer core of the LPS and perturbing the inner core only sensitized the bacteria toward (KFF)3K-PNA conjugates, but not toward conjugates carrying arginine-based BPPs. Interestingly, the chemical composition of the outer LPS core as such, rather than overall hydrophobicity or surface charge, appears to determine the susceptibility to different BPP-PNA conjugates thereby clearly demonstrating the complexity and specificity of the interaction with the LPS/outer membrane. Notably, mutants with outer membrane changes conferring polymyxin resistance did not show resistance toward the BPP-PNA conjugates, thereby eliminating one possible route of resistance for these molecules. Finally, envelope weakening, through deletion of membrane proteins such as OmpA as well as some proteins previously identified as involved in cationic antimicrobial peptide uptake, did not significantly influence BPP-PNA conjugate activity.

Low-Level Cefepime Exposure Induces High-Level Resistance in Environmental Bacteria: Molecular Mechanism and Evolutionary Dynamics.

Antibiotics exert selective pressures on clinically relevant antibiotic resistance. It is critical to understand how antibiotic resistance evolves in environmental microbes exposed to subinhibitory concentrations of antibiotics and whether evolutionary dynamics and emergence of resistance are predictable. In this study, Comamonas testosteroni isolated from wastewater activated sludge were subcultured in a medium containing 10 ng/mL cefepime for 40 days (∼300 generations). Stepwise mutations were accumulated, leading to an ultimate 200-fold increase in the minimum inhibitory concentration (MIC) of cefepime. Early stage mutation in DNA polymerase-encoding gene dnaE2 played an important role in antibiotic resistance evolution. Diverse resistance mechanisms were employed and validated experimentally, including increased efflux, biofilm formation, reduced antibiotic uptake, and drug inactivation. The cefepime minimal selective concentrations (MSCs) and relative fitness of susceptible, intermediate, and resistant mutants were determined. Agent-based modeling of the modified Moran process enabled simulations of resistance evolution and predictions of the emergence time and frequency of resistant mutants. The unraveled cefepime resistance mechanisms could be employed by broader bacteria, and the newly developed model is applicable to the predictions of general resistance evolution. The improved knowledge facilitates the assessment, prediction, and mitigation of antibiotic resistance progression in antibiotic-polluted environments.

The Lysine Acetylation Modification in the Porin Aha1 of Aeromonas hydrophila Regulates the Uptake of Multidrug Antibiotics

Protein lysine acetylation (Kac) modification plays important roles in diverse physiological functions. However, there is little evidence on the role of Kac modification in bacterial antibiotic resistance. Here, we compared the differential expressions of whole-cell proteins and Kac peptides in oxytetracycline sensitive and oxytetracycline resistance (OXYR) strains of Aeromonas hydrophila using quantitative proteomics technologies. We observed a porin family protein Aha1 downregulated in the OXYR strain, which may have an important role in the OXY resistance. Interestingly, seven of eight Kac peptides of Aha1 decreased abundance in OXYR as well. Microbiologic assays showed that the K57R, K187R, and K197R Aha1 mutants significantly increased antibiotic resistance to OXY and reduced the intracellular OXY accumulation in OXY stress. Moreover, these Aha1 mutants displayed multidrug resistance features to tetracyclines and β-lactam antibiotics. The 3D model prediction showed that the Kac states of K57, K187, and K197 sites located at the extracellular pore vestibule of Aha1 may be involved in the uptake of specific types of antibiotics. Overall, our results indicate a novel antibiotic resistance mechanism mediated by Kac modification, which may provide a clue for the development of antibiotic therapy strategies.

Uptake Mechanisms and Regulatory Responses to MECAM- and DOTAM-Based Artificial Siderophores and Their Antibiotic Conjugates in Pseudomonas aeruginosa.

The development of new antibiotics against Gram-negative bacteria has to deal with the low permeability of the outer membrane. This obstacle can be overcome by utilizing siderophore-dependent iron uptake pathways as entrance routes for antibiotic uptake. Iron-chelating siderophores are actively imported by bacteria, and their conjugation to antibiotics allows smuggling the latter into bacterial cells. Synthetic siderophore mimetics based on MECAM (1,3,5-N,N',N″-tris-(2,3-dihydroxybenzoyl)-triaminomethylbenzene) and DOTAM (1,4,7,10-tetrakis(carbamoylmethyl)-1,4,7,10-tetraazacyclododecane) cores, both chelating iron via catechol groups, have been recently applied as versatile carriers of functional cargo. In the present study, we show that MECAM and the MECAM-ampicillin conjugate 3 transport iron into Pseudomonas aeruginosa cells via the catechol-type outer membrane transporters PfeA and PirA and DOTAM solely via PirA. Differential proteomics and quantitative real-time polymerase chain reaction (qRT-PCR) showed that MECAM import induced the expression of pfeA, whereas 3 led to an increase in the expression of pfeA and ampc, a gene conferring ampicillin resistance. The presence of DOTAM did not induce the expression of pirA but upregulated the expression of two zinc transporters (cntO and PA0781), pointing out that bacteria become zinc starved in the presence of this compound. Iron uptake experiments with radioactive 55Fe demonstrated that import of this nutrient by MECAM and DOTAM was as efficient as with the natural siderophore enterobactin. The study provides a functional validation for DOTAM- and MECAM-based artificial siderophore mimetics as vehicles for the delivery of cargo into Gram-negative bacteria.

Occurrence of antibiotics in Lettuce (Lactuca sativa L.) and Radish (Raphanus sativus L.) following organic soil fertilisation under plot-scale conditions: Crop and human health implications

Recent studies have demonstrated the crop uptake of antibiotics (ABs) from soils treated with AB-carrying fertilisers. However, there is a lack of plot-scale studies linking their effects at the agronomic and metabolomic/transcriptomic level to their impact on human health. This paper assesses the plant uptake of 23 ABs following two productive cycles of lettuce and radish cropped with sewage sludge, pig slurry, the organic fraction of municipal solid waste, or chemical fertilisation under plot-scale conditions (32 plots spanning 3-10 m2 each). AB uptake by plants depended on both the vegetable and the AB class and was higher in radish than in lettuce edible parts. Levels ranged from undetectable to up to 76 ng/g (fresh weight). Repetitive organic fertilisation resulted in an increase in the concentration of ABs in lettuce leaves, but not in radish roots. Significant metabolomic and transcriptomic changes were observed following soil fertilisation. Nevertheless, a human health risk assessment indicates that the occurrence of ABs in lettuce or radish edible parts does not pose any risk. To our knowledge, this is the first holistic plot-scale study demonstrating that the use of organic fertilisers containing ABs is safe for crop security and human health.

Scaffold size-dependent effect on the enhanced uptake of antibiotics and other compounds by Escherichia coli and Pseudomonas aeruginosa

The outer membrane of Gram-negative bacteria functions as an impermeable barrier to foreign compounds. Thus, modulating membrane transport can contribute to improving susceptibility to antibiotics and efficiency of bioproduction reactions. In this study, the cellular uptake of hydrophobic and large-scaffold antibiotics and other compounds in Gram-negative bacteria was investigated by modulating the homolog expression of bamB encoding an outer membrane lipoprotein and tolC encoding an outer membrane efflux protein via gene deletion and gene silencing. The potential of deletion mutants for biotechnological applications, such as drug screening and bioproduction, was also demonstrated. Instead of being subjected to gene deletion, wild-type bacterial cells were treated with cell-penetrating peptide conjugates of a peptide nucleic acid (CPP-PNA) against bamB and tolC homologs as antisense agents. Results revealed that the single deletion of bamB and tolC in Escherichia coli increased the uptake of large- and small-scaffold hydrophobic compounds, respectively. A bamB-and-tolC double deletion mutant had a higher uptake efficiency for certain antibiotics and other compounds with high hydrophobicity than each single deletion mutant. The CPP-PNA treated E. coli and Pseudomonas aeruginosa cells showed high sensitivity to various antibiotics. Therefore, these gene deletion and silencing approaches can be utilized in therapeutic and biotechnological fields.

Phytotoxic effects on chloroplast and UHPLC-HRMS based untargeted metabolomic responses in Allium tuberosum Rottler ex Sprengel (Chinese leek) exposed to antibiotics

Introduction of antibiotics into agricultural fields poses serious health risks to humans. This study investigated the uptake of antibiotics, their effects on metabolic pathways, and chloroplast structure changes of Allium tuberosum exposed to norfloxacin (NFL), oxytetracycline (OTC), and tetracycline (TC). Among all the antibiotic treatments, the highest accumulation of antibiotics in roots (4.15 mg/kg) and leaves (0.29 mg/kg) was TC, while in bulbs it was NFL (5.94 mg/kg). OTC was with the lowest accumulation in roots: 0.19 mg/kg, bulbs: 0.18 mg/kg, and leaves: 0.11 mg/kg. The number of mitochondira and the number of plastoglobulli increased. The chloroplast structure was disturbed under the stress of NFL, OTC, and TC. Disturbance in the chloroplast ultrastructure leads to altered chlorophyll fluorescence variables. Simultaneously, metabolomic profiling of leaves demonstrated that NFL stress regulated more of metabolic pathways than OTC and TC. Differences in metabolic pathways among the antibiotic treatments showed that each antibiotic has different impact even under the same experimental conditions. TC and NFL have more toxic effects than OTC antibiotic. Metabolic variations induced by antibiotics stress highlighted pools of metabolites that affect the metabolic activities, chlorophyll fluorescence, ultrastructural adjustments, and stimulate defensive impact in A. tubersoum. These findings provide an insight of metabolic destabilization as well as metabolic changes in defensive mechanism and stress response of A. tuberosum to different antibiotics.

N-Butanol Potentiates Subinhibitory Aminoglycosides against Bacterial Persisters and Multidrug-Resistant MRSA by Rapidly Enhancing Antibiotic Uptake.

Potentiation of traditional antibiotics is of significance for combating antibiotic-resistant bacteria that have become a severe threat to human and animal health. Here, we report that 1 min co-treatment with n-butanol greatly and specifically enhances the bactericidal action of aminoglycosides by 5 orders of magnitude against stationary-phase Staphylococcus aureus cells, with n-propanol and isobutanol showing less potency. This combined treatment also rapidly kills various S. aureus persisters, methicillin-resistant S. aureus (MRSA) cells, and numerous Gram-positive and -negative pathogens including some clinically isolated multidrug-resistant pathogens (e.g., S. aureus, Staphylococcus epidermidis, and Enterococcus faecalis) in vitro, as well as S. aureus in mice. Mechanistically, the potentiation results from the actions of aminoglycosides on their conventional target ribosome rather than the antiseptic effect of n-butanol and is achieved by rapidly enhancing the bacterial uptake of aminoglycosides, while salts and inhibitors of proton motive force (e.g., CCCP) can diminish this uptake. Importantly, such n-butanol-enhanced antibiotic uptake even enables subinhibitory concentrations of aminoglycosides to rapidly kill both MRSA and conventional S. aureus cells. Given n-butanol is a non-metabolite in the pathogens we tested, our work may open avenues to develop a metabolite-independent strategy for aminoglycoside potentiation to rapidly eliminate antibiotic-resistant/tolerant pathogens, as well as for reducing the toxicity associated with aminoglycoside use.

Reducing bacterial antibiotic resistance by targeting bacterial metabolic pathways and disrupting RND efflux pump activity

Antibiotic resistance is a significant issue for the medical community, worldwide. Many bacteria develop drug resistance by utilizing multidrug resistant or MDR efflux pumps that can export antibiotics from bacterial cells. Antibiotics are expelled from bacteria by efflux pumps a part of the resistance nodulation division (RND) family. Types of RND efflux pumps include the AcrAB-TolC tripartite protein pump. There are an excessive number of antibiotic compounds that have been discovered; however, only a few antibiotics are effective against MDR bacteria. Many bacteria become drug resistant when sharing genes that encode MDR efflux pump expression. MDR efflux pump encoding genes are incorporated into plasmids and then shared among bacteria. As a consequence, advancements in genetic engineering can sufficiently target and edit pathogenic bacterial genomes for perturbing drug resistance mechanisms. In this perspective and review, support will be provided for utilizing genetic modifications as an antimicrobial approach and tool that may effectively combat bacterial MDR. Ayhan et al. found that deleting acrB, acrA, and tolC increased the levels of antibiotic sensitivity in Escherichia coli. Researchers also found that glucose, glutamate, and fructose all induced the absorption of antibiotics by upregulating the gene expression of maeA and maeB that is a part of the MAL-pyruvate pathway. Therefore, the current perspective and review will discuss the potential efficacy of reducing antibiotic resistance by inhibiting genes that encode efflux protein pump expression while simultaneously upregulating metabolic genes for increased antibiotic uptake.