Β-lactam Antibiotics Oxacillin Research Articles

Breaking membrane barriers to neutralize E. coli and K. pneumoniae virulence with PEGylated branched polyethylenimine

Bacterial infections caused by Gram-negative pathogens, such as those in the family Enterobacteriaceae, are among the most difficult to treat because effective therapeutic options are either very limited or non-existent. This raises serious concern regarding the emergence and spread of multi-drug resistant (MDR) pathogens in the community setting; and thus, creates the need for discovery efforts and/or early-stage development of novel therapies for infections. Our work is directed towards branched polyethylenimine (BPEI) modified with polyethylene glycol (PEG) as a strategy for targeting virulence from Gram-negative bacterial pathogens. Here, we neutralize lipopolysaccharide (LPS) as a barrier to the influx of antibiotics. Data demonstrate that the β-lactam antibiotic oxacillin, generally regarded as ineffective against Gram-negative bacteria, can be potentiated by 600 Da BPEI to kill some Escherichia coli and some Klebsiella pneumoniae. Modification of 600 Da BPEI with polyethylene glycol (PEG) could increase drug safety and improves potentiation activity. The ability to use the Gram-positive agent, oxacillin, against Gram-negative pathogens could expand the capability to deliver effective treatments that simplify, reduce, or eliminate some complicated treatment regimens.

An Easy-to-Use Plasmid Toolset for Efficient Generation and Benchmarking of Synthetic Small RNAs in Bacteria.

Synthetic biology approaches life from the perspective of an engineer. Standardized and de novo design of genetic parts to subsequently build reproducible and controllable modules, for example, for circuit design, is a key element. To achieve this, natural systems and elements often serve as a blueprint for researchers. Regulation of protein abundance is controlled at DNA, mRNA, and protein levels. Many tools for the activation or repression of transcription or the destabilization of proteins are available, but easy-to-handle minimal regulatory elements on the mRNA level are preferable when translation needs to be modulated. Regulatory RNAs contribute considerably to regulatory networks in all domains of life. In particular, bacteria use small regulatory RNAs (sRNAs) to regulate mRNA translation. Slowly, sRNAs are attracting the interest of using them for broad applications in synthetic biology. Here, we promote a “plug and play” plasmid toolset to quickly and efficiently create synthetic sRNAs to study sRNA biology or their application in bacteria. We propose a simple benchmarking assay by targeting the acrA gene of Escherichia coli and rendering cells sensitive toward the β-lactam antibiotic oxacillin. We further highlight that it may be necessary to test multiple seed regions and sRNA scaffolds to achieve the desired regulatory effect. The described plasmid toolset allows quick construction and testing of various synthetic sRNAs based on the user’s needs.

Impact of FtsZ Inhibition on the Localization of the Penicillin Binding Proteins in Methicillin-Resistant Staphylococcus aureus.

Methicillin-resistant Staphylococcus aureus (MRSA) is a multidrug-resistant pathogen of acute clinical importance. Combination treatment with an FtsZ inhibitor potentiates the activity of penicillin binding protein (PBP)-targeting β-lactam antibiotics against MRSA. To explore the mechanism underlying this synergistic behavior, we examined the impact of treatment with the FtsZ inhibitor TXA707 on the spatial localization of the five PBP proteins expressed in MRSA. In the absence of drug treatment, PBP1, PBP2, PBP3, and PBP4 colocalize with FtsZ at the septum, contributing to new cell wall formation. In contrast, PBP2a localizes to distinct foci along the cell periphery. Upon treatment with TXA707, septum formation becomes disrupted, and FtsZ relocalizes away from midcell. PBP1 and PBP3 remain significantly colocalized with FtsZ, while PBP2, PBP4, and PBP2a localize away from FtsZ to specific sites along the periphery of the enlarged cells. We also examined the impact on PBP2a and PBP2 localization of treatment with β-lactam antibiotic oxacillin alone and in synergistic combination with TXA707. Significantly, PBP2a localizes to the septum in approximately 15% of the oxacillin-treated cells, a behavior that likely contributes to the β-lactam resistance of MRSA. Combination treatment with TXA707 causes both PBP2a and PBP2 to localize in malformed septum-like structures. Our collective results suggest that PBP2, PBP4, and PBP2a may function collaboratively in peripheral cell wall repair and maintenance in response to FtsZ inhibition by TXA707. Cotreatment with oxacillin appears to reduce the availability of PBP2a to assist in this repair, thereby rendering the MRSA cells more susceptible to the β-lactam. IMPORTANCE MRSA is a multidrug-resistant bacterial pathogen of acute clinical importance, infecting many thousands of individuals globally each year. The essential cell division protein FtsZ has been identified as an appealing target for the development of new drugs to combat MRSA infections. Through synergistic actions, FtsZ-targeting agents can sensitize MRSA to antibiotics like the β-lactams that would otherwise be ineffective. This study provides key insights into the mechanism underlying this synergistic behavior as well as MRSA resistance to β-lactam drugs. The results of this work will help guide the identification and optimization of combination drug regimens that can effectively treat MRSA infections and reduce the potential for future resistance.

Combined Efficacy of an Antimicrobial Cationic Peptide Polymer with Conventional Antibiotics to Combat Multidrug-Resistant Pathogens.

Antibiotic-resistant infections are predicted to kill 10 million people worldwide per year by 2050 and to cost the global economy 100 trillion USD. Novel approaches and alternatives to conventional antibiotics are urgently required to combat antimicrobial resistance. We have synthesized a chitosan-based oligolysine antimicrobial peptide, CSM5-K5 (where CSM denotes chitosan monomer repeat units and K denotes lysine amino acid repeat units), that targets multidrug-resistant (MDR) bacterial species. Here, we show that CSM5-K5 exhibits rapid bactericidal activity against methicillin-resistant Staphylococcus aureus (MRSA), MDR Escherichia coli, and vancomycin-resistant Enterococcus faecalis (VRE). Combinatorial therapy of CSM5-K5 with antibiotics to which each organism is otherwise resistant restores sensitivity to the conventional antibiotic. CSM5-K5 alone significantly reduced preformed bacterial biofilm by 2-4 orders of magnitude and, in combination with conventional antibiotics, reduced preformed biofilm by more than 2-3 orders of magnitude at subinhibitory concentrations. Moreover, using a mouse excisional wound infection model, CSM5-K5 treatment reduced bacterial burdens by 1-3 orders of magnitude and acted synergistically with oxacillin, vancomycin, and streptomycin to clear MRSA, VRE, and MDR E.coli, respectively. Importantly, little to no resistance against CSM5-K5 arose for any of the three MDR bacteria during 15 days of serial passage. Furthermore, low level resistance to CSM5-K5 that did arise for MRSA conferred increased susceptibility (collateral sensitivity) to the β-lactam antibiotic oxacillin. This work demonstrates the feasibility and benefits of using this synthetic cationic peptide as an alternative to, or in combination with, traditional antibiotics to treat infections caused by MDR bacteria.

Aspermerodione, a novel fungal metabolite with an unusual 2,6-dioxabicyclo[2.2.1]heptane skeleton, as an inhibitor of penicillin-binding protein 2a

Rising drug resistance limits the treatment options infected by methicillin-resistant Staphylococcus aureus (MRSA). A promising solution for overcoming the resistance of MRSA is to inhibit the penicillin-binding protein 2a (PBP2a). A novel terpene-polyketide hybrid meroterpenoid, aspermerodione (1), characterized by an unusual 2,6-dioxabicyclo[2.2.1]heptane core skeleton, and a new heptacyclic analogue, andiconin C (2), were isolated and identified from the liquid cultures of endophytic fungus Aspergillus sp. TJ23. The structures and their absolute configurations of all chiral centers were elucidated via extensive spectroscopic analyses and electronic circular dichroism (ECD) calculations and determined via single-crystal X-ray diffraction analysis. Aspemerodione (1) was found to be a potential inhibitor of PBP2a, and work synergistically with the β-lactam antibiotics oxacillin and piperacillin against MRSA.

Removal of β-lactam antibiotics from pharmaceutical wastewaters using photo-Fenton process at near-neutral pH.

In this work, the photo-Fenton process at near-neutral pH was applied for the removal of the β-lactam antibiotic oxacillin (OXA) in water using artificial and sunlight. Initially, the main variables of the process (Fe(II), H2O2, and light power) were optimized by a statistical factorial design (23 with center points). The experimental design indicated that 90μmolL-1 of Fe(II), 10mmolL-1 of H2O2, and 30W of power light were the favorable conditions for degradation of OXA at 203μmolL-1. In the photo-Fenton system, the H2O2 alone, UV-light/H2O2, and Fe(II)/H2O2 subsystems presented a significant participation on antibiotic removal. Moreover, based on the primary organic transformation products, a mechanism of OXA degradation was proposed. Under the favorable operational conditions, both the pollutant and the antimicrobial activity were eliminated after 50min of process application. Although at 480min of treatment, only 5% of mineralization was achieved, the level of biodegradability of the solutions increased from 0.08 to 0.98. Interestingly, the presence of pharmaceutical additives (glucose, isopropanol, and oxalic acid) had a moderate interference on the efficiency of the pollutant removal. Additionally, the treatment at pilot scale of the β-lactam antibiotic in a pharmaceutical complex matrix using solar radiation allowed the complete removal of the pollutant and its associated antimicrobial activity in a very short time period (5min). These results evidenced the applicability of the photo-Fenton process to treat wastewaters from pharmaceutical industry loaded with β-lactam antibiotics at near neutral pH values efficiently.

Staphylococcus aureus inactivates daptomycin by releasing membrane phospholipids.

Daptomycin is a bactericidal antibiotic of last resort for serious infections caused by methicillin-resistant Staphylococcus aureus (MRSA)1,2. Although resistance is rare, treatment failure can occur in more than 20% of cases3,4 and so there is a pressing need to identify and mitigate factors that contribute to poor therapeutic outcomes. Here, we show that loss of the Agr quorum-sensing system, which frequently occurs in clinical isolates, enhances S. aureus survival during daptomycin treatment. Wild-type S. aureus was killed rapidly by daptomycin, but Agr-defective mutants survived antibiotic exposure by releasing membrane phospholipids, which bound and inactivated the antibiotic. Although wild-type bacteria also released phospholipid in response to daptomycin, Agr-triggered secretion of small cytolytic toxins, known as phenol soluble modulins, prevented antibiotic inactivation. Phospholipid shedding by S. aureus occurred via an active process and was inhibited by the β-lactam antibiotic oxacillin, which slowed inactivation of daptomycin and enhanced bacterial killing. In conclusion, S. aureus possesses a transient defence mechanism that protects against daptomycin, which can be compromised by Agr-triggered toxin production or an existing therapeutic antibiotic.

Comparative study of the effect of pharmaceutical additives on the elimination of antibiotic activity during the treatment of oxacillin in water by the photo-Fenton, TiO2-photocatalysis and electrochemical processes

Synthetic pharmaceutical effluents loaded with the β-lactam antibiotic oxacillin were treated using advanced oxidation processes (the photo-Fenton system and TiO2 photocatalysis) and chloride mediated electrochemical oxidation (with Ti/IrO2 anodes). Combinations of the antibiotic with excipients (mannitol or tartaric acid), an active ingredient (calcium carbonate, i.e. bicarbonate ions due to the pH) and a cleaning agent (sodium lauryl ether sulfate) were considered. Additionally, urban wastewater that had undergone biological treatment was doped with oxacillin and treated with the tested systems. The evolution of antimicrobial activity was monitored as a parameter of processes efficiency. Although the two advanced oxidation processes (AOPs) differ only in the way they produce OH, marked differences were observed between them. There were also differences between the AOPs and the electrochemical system. Interestingly, each additive had a different effect on each treatment. For water loaded with mannitol, electrochemical treatment was the most suitable option because the additive did not significantly affect the efficiency of the system. Due to the formation of a complex with Fe3+, tartaric acid accelerated the elimination of antibiotic activity during the photo-Fenton process. For TiO2 photocatalysis, the presence of bicarbonate ions contributed to antibiotic activity elimination through the possible formation of carbonate and bicarbonate radicals. Sodium lauryl ether sulfate negatively affected all of the processes. However, due to the higher selectivity of HOCl compared with OH, electrochemical oxidation showed the least inhibited efficiency. For the urban wastewater doped with oxacillin, TiO2 photocatalysis was the most efficient process. These results will help select the most suitable technology for the treatment of water polluted with β-lactam antibiotics.

Degradation of the antibiotic oxacillin in water by anodic oxidation with Ti/IrO2 anodes: Evaluation of degradation routes, organic by-products and effects of water matrix components

In this work the application of electrochemical anodic oxidation for the removal of the β-lactam antibiotic Oxacillin (OXA) was tested. The study was performed using an undivided stirred tank reactor equipped with a DSA (Ti/IrO2) anode and a zirconium spiral cathode. A statistical design of experiments was used to study the influence of different operating variables: substance concentration (6.0–1624.0μmolL−1), current density (3.25–30.25mAcm−2) and NaCl concentration (0.05–0.4molL−1). According to the Pareto chart, current density was the main variable influencing OXA degradation, followed by NaCl concentration. The effect of the initial pH and type of supporting electrolyte was also evaluated. The latter lead to the identification of different degradation routes which depended on the electrolyte employed. By using the best electrolyte and current density, complete OXA removal and total loss of antimicrobial activity was achieved after only 4min of treatment. The efficiency in the oxacillin degradation was not significantly affected by the initial pH (3–9). Interestingly, no mineralization was observed even after long exposure times (8h). However, 70% of the initial chemical oxygen demand (COD) was eliminated and the level of biodegradability (BOD5/COD) increased from 0.03 to 0.84, indicating that the system is able to transform the pollutant into highly oxidized and biodegradable bio-products with no antimicrobial activity. Additionally, different substances and radical scavengers present in wastewaters and natural water did not significantly affect the efficiency of the process. Finally, the more relevant initial aromatic by-products were identified and a degradation schema of the electrochemical oxidation of the OXA antibiotic was proposed.

Reduced expression of PBP-2A by neonatal mecA-positive coagulase-negative staphylococci (CoNS) blood isolates: -lactams are useful first-line agents for the treatment of neonatal CoNS sepsis, restricting the use of vancomycin

Vancomycin use for neonatal coagulase-negative staphylococci (CoNS) sepsis is based on a high CoNS carriage rate of mecA, encoding penicillin-binding protein (PBP)-2a, with low affinity for, and associated with resistance to, β-lactam antibiotics. The relationship between mecA gene carriage, phenotypic expression of the gene by PBP-2a production and in vitro resistance to the β-lactam antibiotics oxacillin, cefazolin and amoxicillin/clavulanate was determined for 85 CoNS blood isolates randomly obtained from our collection of isolates from neonates with CoNS sepsis. The relationship between mecA gene carriage, phenotypic expression of the gene by PBP-2a production and in vitro resistance to the β-lactam antibiotics oxacillin, cefazolin and amoxicillin/clavulanate was determined for randomly obtained CoNS blood isolates from our collection of isolates from neonates with CoNS sepsis. The mecA gene was detected using multiplex PCR, and PBP-2a expression was determined using a latex agglutination (LA) test (Oxoid). β-Lactam susceptibility was determined using the Phoenix automated system and, in addition, by Etest with interpretation of MIC values according to the reference MIC breakpoints adopted from the CLSI guidelines M100-S20, Infobase™. Among 85 CoNS blood isolates, 73 (86%) were mecA positive and 12 (14%) were mecA negative. None of the mecA-negative isolates expressed PBP-2a and all were β-lactam susceptible. All mecA-positive CoNS isolates were oxacillin resistant, although most oxacillin MICs were not very high, ranging from 2 to 8 mg/L for the majority of isolates. Only 8/73 (11%) mecA-positive CoNS isolates had oxacillin MICs ≥32 mg/L (range 32 to >256 mg/L). mecA-positive CoNS blood isolates, although fully resistant to oxacillin, were almost universally susceptible to cefazolin and amoxicillin/clavulanate, which was associated with a low expression rate of PBP-2a. β-Lactam antibiotics are useful for the treatment of neonatal CoNS sepsis, reserving vancomycin for selected cases.

Thioridazine affects transcription of genes involved in cell wall biosynthesis in methicillin-resistant Staphylococcus aureus

The antipsychotic drug thioridazine is a candidate drug for an alternative treatment of infections caused by methicillin-resistant Staphylococcus aureus (MRSA) in combination with the β-lactam antibiotic oxacillin. The drug has been shown to have the capability to resensitize MRSA to oxacillin. We have previously shown that the expression of some resistance genes is abolished after treatment with thioridazine and oxacillin. To further understand the mechanism underlying the reversal of resistance, we tested the expression of genes involved in antibiotic resistance and cell wall biosynthesis in response to thioridazine in combination with oxacillin. We observed that the oxacillin-induced expression of genes belonging to the VraSR regulon is reduced by the addition of thioridazine. The exclusion of such key factors involved in cell wall biosynthesis will most likely lead to a weakened cell wall and affect the ability of the bacteria to sustain oxacillin treatment. Furthermore, we found that thioridazine itself reduces the expression level of selected virulence genes and that selected toxin genes are not induced by thioridazine. In the present study, we find indications that the mechanism underlying reversal of resistance by thioridazine relies on decreased expression of specific genes involved in cell wall biosynthesis.

Restoration of Oxacillin Susceptibility in Methicillin-Resistant Staphylococcus aureus by Blocking the MecR1-Mediated Signaling Pathway

The signal transducing integral membrane protein, MecR1 helps initiate the expression of the antibiotic-resistant gene mecA, which encodes the penicillin-binding protein 2a. MecA participates in the β-lactam resistance of methicillin-resistant Staphylococcus aureus (MRSA). Blocking the MecR1 regulatory pathway may be a novel strategy to combat MRSA. In this study, we introduced an antisense phosphothioate oligodeoxynucleotide (PS-ODN) targeting MecR1 mRNA into the MRSA strain WHO-2, which led to a significant reduction of both MecR1 and PBP2a mRNAs in a concentration-dependent manner. Consequently, the susceptibility of S. aureus WHO-2 to the β-lactam antibiotic oxacillin was restored significantly. Our results indicate that blocking the mecR1-mecI-mecA signaling pathway via an antisense approach might be a viable strategy to restore the susceptibility of MRSA to the existing β-lactam antibiotics.

The tetAB genes of the Corynebacterium striatum R-plasmid pTP10 encode an ABC transporter and confer tetracycline, oxytetracycline and oxacillin resistance in Corynebacterium glutamicum

The tetracycline resistance region of the 50-kb R-plasmid pTP10 from the clinical isolate Corynebacterium striatum M82B was analyzed in Corynebacterium glutamicum ATCC 13032 and confined to a 4.4-kb SphI- SalI DNA fragment. Nucleotide sequence analysis revealed two open reading frames, termed tetA and tetB, specifying proteins of 513 and 528 amino acids, respectively. The deduced amino acid sequences of tetAB displayed similarity to ATP-binding cassette transporters including StrV and StrW of Streptomyces glaucescens which are proposed to play a role in the export of streptomycin-like aminoglycosides. An antibiotic susceptibility screening in C. glutamicum showed that the tetAB genes confer resistance to tetracycline, oxytetracycline and to the structurally and functionally unrelated β-lactam antibiotic oxacillin.