Antibiotic Conjugates Research Articles

CRISPR-AMRtracker: A novel toolkit to monitor the antimicrobial resistance gene transfer in fecal microbiota

The spread of antibiotic resistance genes (ARGs), particularly those carried on plasmids, poses a major risk to global health. However, the extent and frequency of ARGs transfer in microbial communities among human, animal, and environmental sectors is not well understood due to a lack of effective tracking tools. We have developed a novel fluorescent tracing tool, CRISPR-AMRtracker, to study ARG transfer. It combines CRISPR/Cas9 fluorescence tagging, fluorescence-activated cell sorting, 16S rRNA gene sequencing, and microbial community analysis. CRISPR-AMRtracker integrates a fluorescent tag immediately downstream of ARGs, enabling the tracking of ARG transfer without compromising the host cell's antibiotic susceptibility, fitness, conjugation, and transposition. Notably, our experiments demonstrate that sfGFP-tagged plasmid-borne mcr-1 can transfer across diverse bacterial species within fecal samples. This innovative approach holds the potential to illuminate the dynamics of ARG dissemination and provide valuable insights to shape effective strategies in mitigating the escalating threat of antibiotic resistance.

Recent Advances in the Development of Metal/Metal Oxide Nanoparticle and Antibiotic Conjugates (MNP-Antibiotics) to Address Antibiotic Resistance: Review and Perspective.

As per the World Health Organization (WHO), antimicrobial resistance (AMR) is a natural phenomenon whereby microbes develop or acquire genes that render them resistant. The rapid emergence and spread of this phenomenon can be attributed to human activity specifically, the improper and excessive use of antimicrobials for the treatment, prevention, or control of infections in humans, animals, and plants. As a result of this factor, many antibiotics have reduced effectiveness against microbes or may not work fully. Thus, there is a pressing need for the development of new antimicrobial agents in order to counteract antimicrobial resistance. Metallic nanoparticles (MNPs) are well known for their broad antimicrobial properties. Consequently, the use of MNPs with current antibiotics holds significant implications. MNPs, including silver nanoparticles (AgNPS), zinc oxide nanoparticles (ZnONPs), copper nanoparticles (CuNPs), and gold nanoparticles (AuNPs), have been extensively studied in conjunction with antibiotics. However, their mechanism of action is still not completely understood. The interaction between these MNPs and antibiotics can be either synergistic, additive, or antagonistic. The synergistic effect is crucial as it represents the desired outcome that researchers aim for and can be advantageous for the advancement of new antimicrobial agents. This article provides a concise and academic description of the recent advancements in MNP and antibiotic conjugates, including their mechanism of action. It also highlights their possible use in the biomedical field and major challenges associated with the use of MNP-antibiotic conjugates in clinical practice.

Rejuvenation of Meropenem by Conjugation with Tilapia Piscidin-4 Peptide Targeting NDM-1 Escherichia coli.

Gram-negative pathogens that produce β-lactamases pose a serious public health threat as they can render β-lactam antibiotics inactive via hydrolysis. This action contributes to the waning effectiveness of clinical antibiotics and creates an urgent need for new antimicrobials. Antimicrobial peptides (AMPs) exhibiting multimodal functions serve as a potential source in spite of a few limitations. Thus, the conjugation of conventional antibiotics with AMPs may be an effective strategy to leverage the advantages of each component. In this study, we conjugated meropenem to the AMP Tilapia piscidin 4 (TP4) using a typical coupling reaction. The conjugate was characterized by using HPLC-MS, HR-MS, and MS-MS fragmentation analysis. It was then evaluated in terms of antibacterial potency, hemolysis, and cytotoxicity toward RAW264.7 and CCD-966SK cell lines. The conjugation of meropenem with TP4 significantly reduced the cytotoxicity compared to TP4. Conjugation of unprotected TP4 with meropenem resulted in cross-linking at the N-terminal and lysine sites. The structural activity relationship of the two isomers of the TP4-meropenem conjugate was investigated. Both the isomers showed notable antibacterial activities against NDM-1 Escherichia coli and reduced red blood cell hemolysis as compared to TP4. Lysine conjugate (TP4-K-Mero) showed lesser hemolysis than the N-terminal conjugate (TP4-N-Mero). Molecular modeling further revealed that the conjugates can bind to lipopolysaccharides and inhibit NDM-1 β-lactamase. Together, these data show that conjugation of antibiotics with AMP can be a feasible approach to increase the therapeutic profile and effectively target multidrug-resistant pathogens. Furthermore, antibiotic conjugation at different AMP sites tends to show unique biological properties.

Ciprofloxacin Poly(β-amino ester) Conjugates Enhance Antibiofilm Activity and Slow the Development of Resistance.

To tackle the emerging antibiotic resistance crisis, novel antimicrobial approaches are urgently needed. Bacterial biofilms are a particular concern in this context as they are responsible for over 80% of bacterial infections and are inherently more recalcitrant toward antimicrobial treatments. The high tolerance of biofilms to conventional antibiotics has been attributed to several factors, including reduced drug diffusion through the dense exopolymeric matrix and the upregulation of antimicrobial resistance machinery with successful biofilm eradication requiring prolonged high doses of multidrug treatments. A promising approach to tackle bacterial infections involves the use of polymer drug conjugates, shown to improve upon free drug toxicity and bioavailability, enhance drug penetration through the thick biofilm matrix, and evade common resistance mechanisms. In the following study, we conjugated the antibiotic ciprofloxacin (CIP) to a small library of biodegradable and biocompatible poly(β-amino ester) (PBAE) polymers with varying central amine functionality. The suitability of the polymers as antibiotic conjugates was then verified in a series of assays including testing of efficacy and resistance response in planktonic Gram-positive and Gram-negative bacteria and the reduction of viability in mono- and multispecies biofilm models. The most active polymer within the prepared PBAE-CIP library was shown to achieve an over 2-fold increase in the reduction of biofilm viability in a Pseudomonas aeruginosa monospecies biofilm and superior elimination of all the species present within the multispecies biofilm model. Hence, we demonstrate that CIP conjugation to PBAEs can be employed to achieve improved antibiotic efficacy against clinically relevant biofilm models.

A Novel Natural Siderophore Antibiotic Conjugate Reveals a Chemical Approach to Macromolecule Coupling.

Inspired by natural sideromycins, the conjugation of antibiotics to siderophores is an attractive strategy to facilitate "Trojan horse" delivery of antibiotics into bacteria. Genome analysis of a soil bacterium, Dactylosporangium fulvum, found a "hybrid" biosynthetic gene cluster responsible for the production of both an antibiotic, pyridomycin, and a novel chlorocatechol-containing siderophore named chlorodactyloferrin. While both of these natural products were synthesized independently, analysis of the culture supernatant also identified a conjugate of both molecules. We then found that the addition of ferric iron to purified chlorodactyloferrin and pyridomycin instigated their conjugation, leading to the formation of a covalent bond between the siderophore-catechol and the pyridomycin-pyridine groups. Using model reactants, this iron-based reaction was found to proceed through a Michael-type addition reaction, where ferric iron oxidizes the siderophore-catechol group into its quinone form, which is then attacked by the antibiotic pyridyl-nitrogen to form the catechol-pyridinium linkage. These findings prompted us to explore if other "cargo" molecules could be attached to chlorodactyloferrin in a similar manner, and this was indeed confirmed with a pyridine-substituted TAMRA fluorophore as well as with pyridine-substituted penicillin, rifampicin, and norfloxacin antibiotic analogues. The resultant biomimetic conjugates were demonstrated to effectively enter a number of bacteria, with TAMRA-chlorodactyloferrin conjugates causing fluorescent labeling of the bacteria, and with penicillin and rifampicin conjugates eliciting antibiotic activity. These findings open up new opportunities for the design and facile synthesis of a novel class of biomimetic siderophore conjugates with antibiotic activity.

Functionalization of ampicillin and gentamicin with biogenic copper nanoparticles (CuNPs) remodel antimicrobial and cytotoxic outcome against MDR clinical isolates.

The present study reports the functionalization of antibiotic-conjugated Alternanthera pungens and Trichodesma indicum copper nanoparticles (CuNPs). Initially, antibiotic profiling of multi-drug resistant (MDR) clinical isolates against five antibiotics was verified and then gentamicin and ampicillin conjugates of CuNPs were prepared. Biosynthesized nanostructures were characterized through UV-visible spectroscopy, Fourier-transformed infrared spectroscopy, X-ray diffraction and scanning electron microscope. Biogenic synthesized CuNPs displayed highest antibacterial activity (24.0-31.3mm inhibition zones) when capped with gentamicin as compared to the ampicillin-conjugated NPs which showed resistance against most of the bacterial species. A. pungens-derived conjugates of gentamicin (CuAp-GNT) along with the vehicle revealed 4.86 ± 0.20% and 4.25 ± 2.96% hemolytic potential and highest MDA production in S. typhimurium (3.18 ± 1.52µg/mL and 6.31 ± 3.49µg/mL) and K. pneumoniae (2.99 ± 0.90µg/mL and 4.06 ± 1.20µg/mL). Similarly, CuAp-GNT also showed highest DNA protection ability by displaying 1342.99 ± 11.87 band intensity. All-inclusive, CuAp showed more promising effects when conjugated with gentamicin indicating that capping of gentamicin with the active components of the plant-based copper nanostructures increases the antibacterial capacity of the drug. Hence, conjugation of antibiotics with bio-based sources offers great potential for identifying potent drug leads.

Real-Time Impedance-Based Monitoring of the Growth and Inhibition of Osteomyelitis Biofilm Pathogen Staphylococcus aureus Treated with Novel Bisphosphonate-Fluoroquinolone Antimicrobial Conjugates.

Osteomyelitis is a limb- and life-threatening orthopedic infection predominantly caused by Staphylococcus aureus biofilms. Bone infections are extremely challenging to treat clinically. Therefore, we have been designing, synthesizing, and testing novel antibiotic conjugates to target bone infections. This class of conjugates comprises bone-binding bisphosphonates as biochemical vectors for the delivery of antibiotic agents to bone minerals (hydroxyapatite). In the present study, we utilized a real-time impedance-based assay to study the growth of Staphylococcus aureus biofilms over time and to test the antimicrobial efficacy of our novel conjugates on the inhibition of biofilm growth in the presence and absence of hydroxyapatite. We tested early and newer generation quinolone antibiotics (ciprofloxacin, moxifloxacin, sitafloxacin, and nemonoxacin) and several bisphosphonate-conjugated versions of these antibiotics (bisphosphonate-carbamate-sitafloxacin (BCS), bisphosphonate-carbamate-nemonoxacin (BCN), etidronate-carbamate-ciprofloxacin (ECC), and etidronate-carbamate-moxifloxacin (ECX)) and found that they were able to inhibit Staphylococcus aureus biofilms in a dose-dependent manner. Among the conjugates, the greatest antimicrobial efficacy was observed for BCN with an MIC of 1.48 µg/mL. The conjugates demonstrated varying antimicrobial activity depending on the specific antibiotic used for conjugation, the type of bisphosphonate moiety, the chemical conjugation scheme, and the presence or absence of hydroxyapatite. The conjugates designed and tested in this study retained the bone-binding properties of the parent bisphosphonate moiety as confirmed using high-performance liquid chromatography. They also retained the antimicrobial activity of the parent antibiotic in the presence or absence of hydroxyapatite, albeit at lower levels due to the nature of their chemical modification. These findings will aid in the optimization and testing of this novel class of drugs for future applications to pharmacotherapy in osteomyelitis.

Emerging Target-Directed Approaches for the Treatment and Diagnosis of Microbial Infections.

With the rising levels of drug resistance, developing efficient antimicrobial therapies has become a priority. A promising strategy is the conjugation of antibiotics with relevant moieties that can potentiate their activity by target-directing. The conjugation of siderophores with antibiotics allows them to act as Trojan horses by hijacking the microorganisms' highly developed iron transport systems and using them to carry the antibiotic into the cell. Through the analysis of relevant examples of the past decade, this Perspective aims to reveal the potential of siderophore-antibiotic Trojan horses for the treatment of infections and the role of siderophores in diagnostic techniques. Other conjugated molecules will be the subject of discussion, namely those involving vitamin B12, carbohydrates, and amino acids, as well as conjugated compounds targeting protein degradation and β-lactamase activated prodrugs.

Nanoparticles in the Era of Antimicrobial Resistance

Antimicrobial resistance is currently a global crisis and is becoming a major issue for human health. The pharmaceutical industries' efforts to create novel therapeutic choices are being slowed down by the active bacterial resistance to traditional antibiotics. Consequently, the rise in infectious diseases caused by resistant bacteria is accompanied by two main issues. Firstly, there is a rise in antibiotic resistance. Secondly, there is a growing disparity between the discovery of new drugs and the rise in antimicrobial resistance. Different treatment strategies are now being in practice to treat bacterial infections, including targeting bacterial virulence factors, bacteriophage therapy, and alteration of the microbiome. Alternative therapeutic approaches are consequently receiving more consideration as traditional antibiotics grow less effective [1].
 The use of nanoparticles is one of the most promising ways for combating microbial drug resistance. Nitric oxide-releasing nanoparticles (NO NPs), chitosan-containing nanoparticles (chitosan NPs), and metal-containing nanoparticles are a few examples of nanoparticle types that use various ways to simultaneously fight against microorganisms. Nanoparticles can be used to overcome existing drug resistance mechanisms such as decreased drug absorption and increased drug efflux from the microbial cell, biofilm formation, and intrinsic resistance. Finally, antimicrobial drugs can be directed via nanoparticles to the site of infection, enabling greater therapeutic doses to be administered there and overcoming resistance. Antibodies directed against a specific antigen on the surface of the target microorganism can be coupled with nanoparticles. For instance, S. aureus can be killed with great specificity using nanoparticles linked with protein A antibodies [2].
 Nanoparticles' chemical structure enables longer binding, active targeting of antibiotics with surface functionalization at the target location, and defense against enzymes. As a result, reaching a larger antibiotic concentration in the cell eliminates the need for a higher dose, which reduces adverse effects. Antibiotic nanoparticle conjugates have been recognized by several scientists as a novel class of antibacterial drugs that can lessen the issue of multidrug resistance. Numerous nanoparticles with antimicrobial properties, such as silver, gold, zinc oxide, and titanium oxide, have been tested against microbial pathogens that are multidrug-resistant in conjunction with commercially available antibiotics or peptides [3].
 Widespread of infectious diseases caused by multiple resistant bacteria is increased day by day and pose a major threat to public health. Therefore, novel antimicrobial strategies are needed to combat the antimicrobial resistance. To reduce adverse effects, nanoparticles may be a preferable option for treating bacterial infections at very low concentrations.

Sustainable on-farm strategy for the disposal of antibiotic fermentation residue: Co-benefits for resource recovery and resistance mitigation

Antibiotic fermentation residue is a key issue for the sustainable operation of pharmaceutical companies, and its improper disposal may cause antibiotic resistance transfer in the environment. However, little is known about the resource recycling strategy of this pharmaceutical waste. Herein, we used hydrothermal spray-dried (HT+SD) and multi-plate dryer (MD) methods to produce bio-organic fertilizers and applied them to an internal recycling model of a field trial. The concentrations of antibiotics (penicillin, cephalosporin, and erythromycin) in the bio-fertilizer, wastewater, and exhaust gas were in the range of 0.002–0.68 mg/kg, ≤ 0.35 ng/mL, and 0.03–0.89 ng/mL, respectively. The organic matter and total nitrogen, phosphorus, and potassium contents were approximately 80% and 10%, respectively. The soil bacterial community was similar among the fertilizer treatments in the same crop cultivation. A total of 233 antibiotic resistance genes (ARGs) and 43 mobile genetic elements (MGEs) were detected, including seven Rank I ARGs and five Rank II ARGs. Random forest analysis showed that gene acc(3)-Via and plasmid trb-C were biomarkers, for which the resistance and the transfer mechanisms were antibiotic inactivation and conjugation, respectively. The results imply that AFR recycling disposal mode is a promising prospect for pharmaceutical waste management.

Silver Nanoparticles Conjugated with Colistin Enhanced the Antimicrobial Activity against Gram-Negative Bacteria.

Colistin is a potent peptide antibiotic that is effective against Gram-negative bacteria. However, nephrotoxicity limited its clinical use. Silver nanoparticles (AgNPs) have gained attention as a potential antimicrobial agent and nanodrug carrier. The conjugation of antibiotics and AgNPs has been found to increase the activity and decrease drug toxicity. In this study, colistin was conjugated with AgNPs (Col-AgNPs), which was confirmed by Fourier-transform infrared (FT-IR) and energy-dispersive X-ray (EDX) spectra. The optimized Col-AgNPs had the proper characteristics, including spherical shape, monodispersity, nanosized particle, high surface charge, and good stability. The powder X-ray diffraction (PXRD) pattern supported the crystallinity of Col-AgNPs and AgNPs. The drug loading of Col-AgNPs was 11.55 ± 0.93%. Col-AgNPs had higher activity against Gram-negative bacteria (Escherichia coli, Klebsiella pneumonia, and Pseudomonas aeruginosa) than AgNPs and colistin. The mechanism of actions of Col-AgNPs involved membrane disruption and genomic DNA damage. The Col-AgNPs and AgNPs were biocompatible with human red blood cells and renal cells at concentrations up to 16 µg/mL. Interestingly, Col-AgNPs exhibited higher cell survival than AgNPs and colistin at 32 µg/mL. Our results revealed that the Col-AgNPs could enhance the antimicrobial activity and cell biocompatibility more than colistin and AgNPs.

Characterization of IncI1/ST71 and IncF18:A−:B1 multidrug-resistance plasmids from an avian Escherichia coli isolate

To characterize IncI1 and IncF18:A−:B1 multidrug-resistance plasmids from an avian Escherichia coli isolate, antibiotic susceptibility testing, conjugation assays, transformation assays, S1-PFGE, and WGS analysis were performed. The 119,457-bp plasmid pEC014–1 with a multidrug-resistance region (MRR) containing four different segments interspersed with six IS26 elements, belonged to incompatibility group I1 and sequence type 71. The 154,516-bp plasmid pEC014–2 with two replicons, typed as FII-18 and FIB-1, carried 14 resistance determinants including blaTEM-1b, blaOXA-1, oqxAB, dfrA17, aac(6′)-Ib-cr, sul1, sul2, tet(A), floR, catB3, hph(aph(4)-Ia), aacC4(aac(3)-IV), aadA5, arr-3, and a merEDACPTR loci in MRR, and additionally encoded three virulence loci: iroNEDCB, sitABCD, and iucABCD-iutA. Plasmid stability assays showed that pEC014–1 and pEC014–2 were stable in recipient E. coli C600 for at least 15 days of passage. Competition assays were carried out to evaluate the fitness impact of pEC014–2 carriage in vitro, revealing a decrease in host fitness. Growth kinetics showed that the growth rate for pEC014–1 or/and pEC014–2 bearing cells was significantly slower than that of the E. coli C600 host strain in the exponential stage (p < 0.01), with only cells carrying pEC014–1 sustaining rapid growth after 6 h of exponential growth. Our findings highlight the mosaic structures of epidemic plasmid IncI1/ST71 and F18:A−:B1 lineages and contribute to a better understanding of the evolution and dissemination of these multidrug resistance and virulence plasmids.

Novel Therapeutics Involving Antibiotic Polymer Conjugates for Treating Various Ailments: A Review.

Antibiotic polymer conjugates (APCs) are an essential part of polymer therapeutics. These conjugates have been used as an appealing platform for drug delivery. As a delivery vector, the administration route severely impacts the accessibility of antibiotics to their respective target site and therapeutic index. Furthermore, the physicochemical and biological properties of conjugates also correlate distinctly with the route of administration. The APCs delivery methods that have been disclosed so far suffer from significant constraints due to poor technology and constrained administration routes (mainly injections). Leading to promising directions, which include the development of specific characteristics for each polymer carrier, application of novel biodegradable polymers, expansion of traditional drug administration routes through the development of emerging routes, and the development of a rational and systematic methodology for designing administration routes are yet to be explored widely. This review focuses primarily on recent improvements in various routes of administration (dental, topical, and ocular) employing APCs. The mechanism of action, as well as other perspectives, have also been discussed. Moreover, this innovative technology provides a fresh perspective on pharmaceutical science research and offers unique and potential pathways for designing desired APCs.

Emergence of a High-Risk Klebsiella michiganensis Clone Disseminating Carbapenemase Genes.

Klebsiella michiganensis is emerging as an important human pathogen of concern especially strains with plasmid-mediated carbapenemase genes. The IncX3-blaNDM-5 plasmid has been described as the primary vector for blaNDM-5 dissemination. However, whether strains with this plasmid have any competitive edge remain largely unexplored. We characterized a blaNDM-5-producing Klebsiella michiganensis strain (KO_408) from Japan and sought to understand the driving force behind the recent dissemination of IncX3-blaNDM-5 plasmids in different bacterial hosts. Antibiotic susceptibility testing, conjugation, and whole-genome sequencing were performed for KO_408, a clinical isolate recovered from a respiratory culture. Fitness, stability, and competitive assays were performed using the IncX3-blaNDM-5 plasmid, pKO_4-NDM-5. KO_408 was ascribed to a novel sequence type, ST256, and harbored resistance genes conforming to its MDR phenotype. The blaNDM-5 gene was localized on the ~44.9 kb IncX3 plasmid (pKO_4-NDM-5), which was transferable in the conjugal assay. The acquisition of pKO_4-NDM-5 did not impose any fitness burden and showed high stability in the host cells. However, transformants with pKO_4-NDM-5 were outcompeted by their host cells and transconjugants with the IncX3-blaOXA-181 plasmid. The genetic environment of blaNDM-5 in pKO_4-NDM-5 has been previously described. pKO_4-NDM-5 showed a close phylogenetic distance with seven similar plasmids from China. KO_408 clustered with strains within the KoI phylogroup, which is closely associated with carbapenemase genes. This study highlights the emergence of a high-risk Klebsiella michiganensis clone harboring carbapenemase genes and affirms that the recent spread of IncX3-blaNDM-5 plasmids might be due to their low fitness cost and stability but not their competitive prowess.

Tracking Uptake and Metabolism of Xenometallomycins Using a Multi-Isotope Tagging Strategy.

Synthetic and naturally occurring siderophores and their conjugates provide access to the bacterial cytoplasm via active membrane transport. Previously, we displaced iron with the radioactive isotope 67Ga to quantify and track in vitro and in vivo uptake and distribution of siderophore Trojan Horse antibiotic conjugates. Here, we introduce a multi-isotope tagging strategy to individually elucidate the fate of metal cargo and the ligand construct with radioisotopes 67Ga and 124I. We synthesized gallium(III) model complexes of a ciprofloxacin-functionalized linear desferrichrome (Ga-D6) and deferoxamine (Ga-D7) incorporating an iodo-tyrosine linker to enable radiolabeling using the metal-binding (67Ga) and the cargo-conjugation site (124I). Radiochemical experiments with Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa wt strains show that 67Ga-D6/D7 and Ga-D6-124I/D7-124I have comparable uptake, indicating intact complex import and siderophore-mediated uptake. In naive mice, 67Ga-D6/D7 and Ga-D6-124I/D7-124I demonstrate predominantly renal clearance; urine metabolite analysis indicates in vivo dissociation of Ga(III) is a likely mechanism of degradation for 67Ga-D6/D7 when compared to ligand radiolabeled compounds, Ga-D6-124I/D7-124I, which remain >60% intact in urine. Cumulatively, this work demonstrates that a multi-isotope tagging strategy effectively elucidates the in vitro uptake, pharmacokinetics, and in vivo stability of xenometallomycins with modular chemical structures.

Mechanical characterization and adhesive properties of a dental adhesive modified with a polymer antibiotic conjugate

This study is a follow up investigation on recent work by our group demonstrating synthesis, release and strong antibacterial character of resins modified with penicillin V (PV)-based polymer-antibiotic conjugates (PACs). Here, we aimed to evaluate the mechanical, bonding, and other relevant biomedical properties of a commercial adhesive resin modified with PV-PAC. Single Bond Plus (SB+) was modified with PAC containing 1.8 wt% conjugated PV. Adhesive resins were bonded to dentin from extracted human molars and restorative resin added. Beams of cross-sectional area of 0.9 ± 0.1 mm (Kutsch and Young, 2011) (n = 20) were obtained from the molars and tested for micro-tensile bond strength (μTBS) at 24 h and 4 months. For cohesive strength, hourglass beams (10 × 2 × 1 mm; n = 10) were assessed for ultimate tensile strength (UTS), beam-shaped specimens (25x2x2 mm; n = 10) evaluated for flexural strength and modulus (FS/FM) via three-point bending, and cylindrical specimens (3 × 2 mm; n = 10) assessed for ultimate compressive strength (UCS). For surface micro-hardness (MH), cylindrical specimens (3 × 2 mm; n = 6) were assessed before and after an EtOH challenge. The degree of conversion (DC) (5 × 1 mm; n = 6) was determined based on changes in absorbance ratio between peaks at ∼1637 cm-1 and ∼1608 cm-1 before and after curing of adhesive resins using attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy. To monitor water uptake and diffusion kinetics over a 28-day period, specimens (5 × 1mm) were desiccated, weighed and stored in deionized water. Control and PV-PAC modified adhesive resins demonstrated similar μTBS at 24 h and 4 months; both showing decrease in values after 4 months (p = 0.001 and 0.004). No significant differences between adhesive resins were shown in UTS, FS/FM or UCS (p<0.05). MH of PV-PAC adhesive resin was significantly reduced relative to the control (p<0.001). The DC values of the adhesive resins were not significantly different. While sorption and solubility were no different between materials, the diffusion coefficient of PV-PAC modified adhesive resin was higher than the control (p<0.001). We conclude that incorporation of PV-PAC with 1.8 wt% PV into an adhesive resin does not adversely affect its mechanical, bonding, and physical properties, thus providing a promising option for materials with long-term antibacterial character and on-demand release.

ESBL Producing Escherichia coli in Faecal Sludge Treatment Plants: An Invisible Threat to Public Health in Rohingya Camps, Cox's Bazar, Bangladesh.

Introduction: Human faecal sludge contains diverse harmful microorganisms, making it hazardous to the environment and public health if it is discharged untreated. Faecal sludge is one of the major sources of E. coli that can produce extended-spectrum β-lactamases (ESBLs).Objective: This study aimed to investigate the prevalence and molecular characterization of ESBL-producing E. coli in faecal sludge samples collected from faecal sludge treatment plants (FSTPs) in Rohingya camps, Bangladesh.Methods: ESBL producing E. coli were screened by cultural as well as molecular methods and further characterized for their major ESBL genes, plasmid profiles, pathotypes, antibiotic resistance patterns, conjugation ability, and genetic similarity.Results: Of 296 isolates, 180 were phenotypically positive for ESBL. All the isolates, except one, contained at least one ESBL gene that was tested (blaCTX−M−1, blaCTX−M−2, blaCTX−M−8, blaCTX−M−9, blaCTX−M−15, blaCTX−M−25, blaTEM, and blaSHV). From plasmid profiling, it was observed that plasmids of 1–211 MDa were found in 84% (151/180) of the isolates. Besides, 13% (24/180) of the isolates possessed diarrhoeagenic virulence genes. From the remaining isolates, around 51% (79/156) harbored at least one virulence gene that is associated with the extraintestinal pathogenicity of E. coli. Moreover, 4% (3/156) of the isolates were detected to be potential extraintestinal pathogenic E. coli (ExPEC) strains. Additionally, all the diarrhoeagenic and ExPEC strains showed resistance to three or more antibiotic groups which indicate their multidrug-resistant potential. ERIC-PCR differentiated these pathogenic isolates into seven clusters. In addition to this, 16 out of 35 tested isolates transferred plasmids of 32–112 MDa to E. coli J53 recipient strain.Conclusion: The present study implies that the faecal sludge samples examined here could be a potential origin for spreading MDR pathogenic ESBL-producing E. coli. The exposure of Rohingya individuals, living in overcrowded camps, to these organisms poses a severe threat to their health.

Nonclinical toxicology development of a novel antibody antibiotic conjugate for treating invasive Staphylococcus Aureus infections

Invasive Staphylococcus aureus (S. aureus) infections are a leading cause of death and not effectively treated with prolonged standard of care antibiotics. A novel THIOMAB™ antibody antibiotic conjugate (TAC) was developed that uses a bacterial-wall specific antibody to deliver the antibiotic (dmDNA31, a rifamycin analogue) to bacteria to minimize toxicities typically seen with prolonged use of traditional antibiotics. The TAC nonclinical toxicology package included repeat dose rat and cynomolgus monkey toxicology studies for 8 weekly intravenous (IV) doses, a 7-day daily repeat dose IV toxicology study of dmDNA31 and an assessment of genotoxicity, cardiovascular toxicity, neurotoxicity and sperm parameters. TAC and dmDNA31 were well tolerated in rats and monkeys, and there was no evidence of genotoxicity, cardiovascular toxicity or neurotoxicity. Non-adverse findings were observed and included blue discoloration in skin, blood, etc. due to the blue color of dmDNA31, increased globulin due to the high doses of antibodies, and abnormal sperm morphology of small heads in male rats with no histopathology correlate in testis. This is an example of antibody-mediated delivery of an antibiotic that has the potential to offer a more effective way of eradicating infection while providing a better safety profile compared to traditional antibiotics.

Antimicrobial Activity of Nanoconjugated Glycopeptide Antibiotics and Their Effect on Staphylococcus aureus Biofilm.

In the era of antimicrobial resistance, the use of nanoconjugated antibiotics is regarded as a promising approach for preventing and fighting infections caused by resistant bacteria, including those exacerbated by the formation of difficult-to-treat bacterial biofilms. Thanks to their biocompatibility and magnetic properties, iron oxide nanoparticles (IONPs) are particularly attractive as antibiotic carriers for the targeting therapy. IONPs can direct conjugated antibiotics to infection sites by the use of an external magnet, facilitating tissue penetration and disturbing biofilm formation. As a consequence of antibiotic localization, a decrease in its administration dosage might be possible, reducing the side effects to non-targeted organs and the risk of antibiotic resistance spread in the commensal microbiota. Here, we prepared nanoformulations of the ‘last-resort’ glycopeptides teicoplanin and vancomycin by conjugating them to IONPs via surface functionalization with (3-aminopropyl) triethoxysilane (APTES). These superparamagnetic NP-TEICO and NP-VANCO were chemically stable and NP-TEICO (better than NP-VANCO) conserved the typical spectrum of antimicrobial activity of glycopeptide antibiotics, being effective against a panel of staphylococci and enterococci, including clinical isolates and resistant strains. By a combination of different methodological approaches, we proved that NP-TEICO and, although to a lesser extent, NP-VANCO were effective in reducing biofilm formation by three methicillin-sensitive or resistant Staphylococcus aureus strains. Moreover, when attracted and concentrated by the action of an external magnet, NP-TEICO exerted a localized inhibitory effect on S. aureus biofilm formation at low antibiotic concentration. Finally, we proved that the conjugation of glycopeptide antibiotics to IONPs reduced their intrinsic cytotoxicity toward a human cell line.

Thermoresponsive Polymer-Antibiotic Conjugates Based on Gradient Copolymers of 2-Oxazoline and 2-Oxazine.

A polymer-antibiotic conjugate with thermoresponsive properties near body temperature is presented. The backbone polymer is a copolymer of 2-n-propyl-2-oxazine (PropOzi) and methoxycarbonylethyl-2-oxazoline (C2MestOx) which is conjugated with the broad-spectrum antibiotic, cefazolin, via modification of the methyl ester group of C2MestOx. The resulting polymer-antibiotic conjugate has a cloud point temperature near body temperature, meaning that it can form a homogenous solution if cooled, but when injected into a skin-mimic at 37 °C, it forms a drug depot precipitate. Cleavage of the ester linker leads to quantitative release of the pristine cefazolin (with some antibiotic degradation observed) and redissolution of the polymer. When Escherichia coli were treated with polymer-antibiotic conjugate total clearance is observed within 12 h. The power of this approach is the potential for localized antibiotic delivery, for example, at a specific tissue site or into infected phagocytic cells.