Analysis of molecular epidemiological characteristics and antimicrobial susceptibility of vancomycin-resistant and linezolid-resistant Enterococcus in China.

BACKGROUND: This study investigates the distribution and characteristics of linezolid and vancomycin susceptibilities among Enterococcus faecalis (E. faecalis) and Enterococcus faecium (E. faecium) and explores the underlying resistance mechanisms. METHODS: A total of 2842 Enterococcus clinical isolates from patients were retrospectively collected, and their clinical data were further analyzed. The minimum inhibitory concentrations (MICs) of vancomycin and linezolid were validated by broth dilution method. The resistance genes optrA, cfr, vanA, vanB and vanM were investigated using polymerase chain reaction (PCR). Housekeeping genes and resistance genes were obtianed through whole-genome sequencing (WGS). RESULTS: Of the 2842 Enterococcus isolates, 88.5% (2516) originated from urine, with E. faecium accounted for 60.1% of these. The vanA gene was identified in 27/28 vancomycin resistant Enterococcus (VRE) isolates, 4 of which carried both vanA and vanM genes. The remaining strain was vanM positive. The optrA gene was identified in all E. faecalis isolates among linezolid resistant Enterococcus (LRE). E. faecium showed a higher multiple antibiotic resistance index (MAR index) compared to E. faecalis. The multi-locus sequence typing (MLST) showed the sequence type of E. faecium mainly belongs to clonal complex (CC) 17, nearly E. faecalis isolates analyzed were differentiated into 7 characteristics of sequence types (STs), among which ST16 of CC16 were the major lineage. CONCLUSION: Urine was the primary source of VRE and LRE isolates in this study. E. faecium showed higher levels of resistance compared to E. faecalis. OptrA gene was detected in 91.6% of LRE, which could explain linezolid resistance, and van genes were detected in all vancomycin resistant Enterococcus strains, while vanA was a key resistance mechanism in VRE identified in this study.

Optimizing vancomycin therapeutic drug monitoring compliance in pediatric oncology: towards personalized medication management.

Aim: Vancomycin, a crucial treatment for Gram-positive bacteria, necessitates therapeutic drug monitoring (TDM) to prevent treatment failures. We investigated the healthcare professional's compliance toward TDM of vancomycin recommendations and follow-up levels. Materials & methods: We collected data from 485 patients who received vancomycin in the Children's Cancer Hospital Egypt 57357 medical records system (Cerner) over 4 months, from January to April 2020. Results: Our data shows that only 54% of patients had TDM requests from healthcare professionals for the total patients who received vancomycin treatment. The healthcare professionals' compliance with the recommendations was 91.7%, while the follow-up levels were 66.7%. Conclusion: While overall adherence to recommendations is strong, enhancing compliance with follow-up levels remains a priority for improvement.

[Box: see text].

Detection of a Kinetically Competent Compound-I Intermediate in the Vancomycin Biosynthetic Enzyme OxyB.

Cytochrome P450 enzymes are abundantly encoded in microbial genomes. Their reactions have two general outcomes, one involving oxygen insertion via a canonical "oxygen rebound" mechanism and a second that diverts from this pathway and leads to a wide array of products, notably intramolecular oxidative cross-links. The antibiotic of-last-resort, vancomycin, contains three such cross-links, which are crucial for biological activity and are installed by the P450 enzymes OxyB, OxyA, and OxyC. The mechanisms of these enzymes have remained elusive in part because of the difficulty in spectroscopically capturing transient intermediates. Using stopped-flow UV/visible absorption and rapid freeze-quench electron paramagnetic resonance spectroscopies, we show that OxyB generates the highly reactive compound-I intermediate, which can react with a model vancomycin peptide substrate in a kinetically competent fashion to generate product. Our results have implications for the mechanism of OxyB and are in line with the notion that oxygen rebound and oxidative cross-links share early steps in their catalytic cycles.

Development and Validation of Liquid Chromatographic Method for Fast Determination of Lincomycin, Polymyxin and Vancomycin in Preservation Solution for Transplants.

In this study, a liquid chromatographic method was developed for the fast determination of lincomycin, polymyxin and vancomycin in a preservation solution for transplants. A Kinetex EVO C18 (150 × 4.6 mm, 2.6 µm) column was utilized at 45 °C. Gradient elution was applied using a mixture of mobile phases A and B, both including 30 mM phosphate buffer at pH 2.0 and acetonitrile, at a ratio of 95:5 (v/v) for A and 50:50 (v/v) for B. A flow rate of 1.0 mL/min, an injection volume of 20 µL and UV detection at 210 nm were used. A degradation study treating the three antibiotics with 0.5 M hydrochloric acid, 0.5 M sodium hydroxide and 3% H2O2 indicated that the developed method was selective toward lincomycin, polymyxin, vancomycin and their degradation products. Other ingredients of the preservation solution, like those from the cell culture medium, did not interfere. The method was validated with good sensitivity, linearity, precision and accuracy. Furthermore, lincomycin, polymyxin and vancomycin were found to be stable in this preservation solution for 4 weeks when stored at -20 °C.

Antibacterial and antibiofilm potentials of vancomycin-loaded niosomal drug delivery system against methicillin-resistant Staphylococcus aureus (MRSA) infections.

The threat of methicillin-resistant Staphylococcus aureus (MRSA) is increasing worldwide, making it significantly necessary to discover a novel way of dealing with related infections. The quick spread of MRSA isolates among infected individuals has heightened public health concerns and significantly limited treatment options. Vancomycin (VAN) can be applied to treat severe MRSA infections, and the indiscriminate administration of this antimicrobial agent has caused several concerns in medical settings. Owing to several advantageous characteristics, a niosomal drug delivery system may increase the potential of loaded antimicrobial agents. This work aims to examine the antibacterial and anti-biofilm properties of VAN-niosome against MRSA clinical isolates with emphasis on cytotoxicity and stability studies. Furthermore, we aim to suggest an effective approach against MRSA infections by investigating the inhibitory effect of formulated niosome on the expression of the biofilm-associated gene (icaR). The thin-film hydration approach was used to prepare the niosome (Tween 60, Span 60, and cholesterol), and field emission scanning electron microscopy (FE-SEM), an in vitro drug release, dynamic light scattering (DLS), and entrapment efficiency (EE%) were used to investigate the physicochemical properties. The physical stability of VAN-niosome, including hydrodynamic size, polydispersity index (PDI), and EE%, was analyzed for a 30-day storage time at 4 °C and 25 °C. In addition, the human foreskin fibroblast (HFF) cell line was used to evaluate the cytotoxic effect of synthesized niosome. Moreover, minimum inhibitory and bactericidal concentrations (MICs/MBCs) were applied to assess the antibacterial properties of niosomal VAN formulation. Also, the antibiofilm potential of VAN-niosome was investigated by microtiter plate (MTP) and real-time PCR methods. The FE-SEM result revealed that synthesized VAN-niosome had a spherical morphology. The hydrodynamic size and PDI of VAN-niosome reported by the DLS method were 201.2 nm and 0.301, respectively. Also, the surface zeta charge of the prepared niosome was - 35.4 mV, and the EE% ranged between 58.9 and 62.5%. Moreover, in vitro release study revealed a sustained-release profile for synthesized niosomal formulation. Our study showed that VAN-niosome had acceptable stability during a 30-day storage time. Additionally, the VAN-niosome had stronger antibacterial and anti-biofilm properties against MRSA clinical isolates compared with free VAN. In conclusion, the result of our study demonstrated that niosomal VAN could be promising as a successful drug delivery system due to sustained drug release, negligible toxicity, and high encapsulation capacity. Also, the antibacterial and anti-biofilm studies showed the high capacity of VAN-niosome against MRSA clinical isolates. Furthermore, the results of real-time PCR exhibited that VAN-niosome could be proposed as a powerful strategy against MRSA biofilm via down-regulation of icaR gene expression.

Fitness costs of Tn1546-type transposons harboring the vanA operon by plasmid type and structural diversity in Enterococcus faecium.

BACKGROUND: This study analyzed the genetic traits and fitness costs of vancomycin-resistant Enterococcus faecium (VREfm) blood isolates carrying Tn1546-type transposons harboring the vanA operon. METHODS: All E. faecium blood isolates were collected from eight general hospitals in South Korea during one-year study period. Antimicrobial susceptibility testing and vanA and vanB PCR were performed. Growth rates of E. faecium isolates were determined. The vanA-positive isolates were subjected to whole genome sequencing and conjugation experiments. RESULTS: Among 308 E. faecium isolates, 132 (42.9%) were positive for vanA. All Tn1546-type transposons harboring the vanA operon located on the plasmids, but on the chromosome in seven isolates. The plasmids harboring the vanA operon were grouped into four types; two types of circular, nonconjugative plasmids (Type A, n = 50; Type B, n = 46), and two types of putative linear, conjugative plasmids (Type C, n = 16; Type D, n = 5). Growth rates of vanA-positive E. faecium isolates were significantly lower than those of vanA-negative isolates (P < 0.001), and reduction in growth rate under vancomycin pressure was significantly larger in isolates harboring putative linear plasmids than in those harboring circular plasmids (P = 0.020). CONCLUSIONS: The possession of vanA operon was costly to bacterial hosts in antimicrobial-free environment, which provide evidence for the importance of reducing vancomycin pressure for prevention of VREfm dissemination. Fitness burden to bacterial hosts was varied by type and size of the vanA operon-harboring plasmid.

Genome assembly, comparative genomics, and identification of genes/pathways underlying plant growth-promoting traits of an actinobacterial strain, Amycolatopsis sp. (BCA-696).

The draft genome sequence of an agriculturally important actinobacterial species Amycolatopsis sp. BCA-696 was developed and characterized in this study. Amycolatopsis BCA-696 is known for its biocontrol properties against charcoal rot and also for plant growth-promotion (PGP) in several crop species. The next-generation sequencing (NGS)-based draft genome of Amycolatopsis sp. BCA-696 comprised of ~ 9.05 Mb linear chromosome with 68.75% GC content. In total, 8716 protein-coding sequences and 61 RNA-coding sequences were predicted in the genome. This newly developed genome sequence has been also characterized for biosynthetic gene clusters (BGCs) and biosynthetic pathways. Furthermore, we have also reported that the Amycolatopsis sp. BCA-696 produces the glycopeptide antibiotic vancomycin that inhibits the growth of pathogenic gram-positive bacteria. A comparative analysis of the BCA-696 genome with publicly available closely related genomes of 14 strains of Amycolatopsis has also been conducted. The comparative analysis has identified a total of 4733 core and 466 unique orthologous genes present in the BCA-696 genome The unique genes present in BCA-696 was enriched with antibiotic biosynthesis and resistance functions. Genome assembly of the BCA-696 has also provided genes involved in key pathways related to PGP and biocontrol traits such as siderophores, chitinase, and cellulase production.

Comparison of the effect of combined usage of povidone-iodine irrigation and topical vancomycin powder to the use of povidone-iodine irrigation alone on the periprosthetic joint infection incidence rate in patients undergoing primary total hip and knee arthroplasty: a protocol for multicenter prospective randomized clinical trial.

BACKGROUND: With the increasing number of joint replacement surgeries, periprosthetic joint infection (PJI) has become a significant concern in orthopedic practice, making research on PJI prevention paramount. Therefore, the study will aim to compare the effect of combined usage of povidone-iodine and topical vancomycin powder to the use of povidone-iodine alone on the PJI incidence rate in patients undergoing primary total hip (THA) and total knee arthroplasty (TKA). METHODS: The prospective randomized clinical trial will be conducted in two independent voivodeship hospitals with extensive experience in lower limb arthroplasties. The studied material will comprise 840 patients referred to hospitals for primary THA or TKA. The patients will be randomly allocated to two equal groups, receiving two different interventions during joint replacement. In group I, povidone-iodine irrigation and consecutively topical vancomycin powder will be used before wound closure. In group II, only povidone-iodine lavage irrigation will be used before wound closure. The primary outcome will be the incidence rate of PJI based on the number of patients with PJI occurrence within 90 days after arthroplasty. The occurrence will be determined using a combined approach, including reviewing hospital records for readmissions and follow-up phone interviews with patients. The infection will be diagnosed based on Musculoskeletal Infection Society criteria. The chi-square test will be used to compare the infection rates between the two studied groups. Risk and odds ratios for the between-groups comparison purposes will also be estimated. Medical cost analysis will also be performed. DISCUSSION: A randomized clinical trial comparing the effect of combined usage of povidone-iodine irrigation and vancomycin powder to the use of povidone-iodine irrigation alone in preventing PJIs after primary arthroplasty is crucial to advancing knowledge in orthopedic surgery, improving patient outcomes, and guiding evidence-based clinical practices. TRIAL REGISTRATION: ClinicalTrials.gov NCT05972603 . Registered on 2 August 2023.

Dual-recognition colorimetric platform based on porous Au@Pt nanozymes for highly sensitive washing-free detection of Staphylococcus aureus.

A dual-recognition strategy is reported to construct a one-step washing and highly efficient signal-transduction tag system for high-sensitivity colorimetric detection of Staphylococcus aureus (S. aureus). The porous (gold core)@(platinum shell) nanozymes (Au@PtNEs) as the signal labels show highly efficient peroxidase mimetic activity and are robust. For the sake of simplicity the detection involved the use of a vancomycin-immobilized magnetic bead (MB) and aptamer-functionalized Au@PtNEs for dual-recognition detection in the presence of S. aureus. In addition, we designed a magnetic plate to fit the 96-well microplate to ensure consistent magnetic properties of each well, which can quickly remove unreacted Au@PtNEs and sample matrix while avoiding tedious washing steps. Subsequently, Au@PtNEs catalyze hydrogen peroxide (H2O2) to oxidize 3,3',5,5'-tetramethylbenzidine (TMB) generating a color signal. Finally, the developed Au@PtNEs-based dual-recognition washing-free colorimetric assay displayed a response in the range of S. aureus of 5 × 101-5 × 105 CFU/mL, and the detection limit was 40 CFU/mL within 1.5 h. In addition, S. aureus-fortified samples were analyzed to further evaluate the performance of the proposed method, which yielded average recoveries ranging from 93.66 to 112.44% and coefficients of variation (CVs) within the range 2.72-9.01%. These results furnish a novel horizon for the exploitation of a different mode of recognition and inexpensive enzyme-free assay platforms as an alternative to traditional enzyme-based immunoassays for the detection of other Gram-positive pathogenic bacteria.

A classic antibiotic reimagined: Rationally designed bacitracin variants exhibit potent activity against vancomycin-resistant pathogens.

Bacitracin is a macrocyclic peptide antibiotic that is widely used as a topical treatment for infections caused by gram-positive bacteria. Mechanistically, bacitracin targets bacteria by specifically binding to the phospholipid undecaprenyl pyrophosphate (C55PP), which plays a key role in the bacterial lipid II cycle. Recent crystallographic studies have shown that when bound to C55PP, bacitracin adopts a highly ordered amphipathic conformation. In doing so, all hydrophobic side chains align on one face of the bacitracin-C55PP complex, presumably interacting with the bacterial cell membrane. These insights led us to undertake structure-activity investigations into the individual contribution of the nonpolar amino acids found in bacitracin. To achieve this we designed, synthesized, and evaluated a series of bacitracin analogues, a number of which were found to exhibit significantly enhanced antibacterial activity against clinically relevant, drug-resistant pathogens. As for the natural product, these next-generation bacitracins were found to form stable complexes with C55PP. The structure-activity insights thus obtained serve to inform the design of C55PP-targeting antibiotics, a key and underexploited antibacterial strategy.

Suspensions of antibiotics in self-emulsifying oils as a novel approach to formulate eye drops with substances which undergo hydrolysis in aqueous environment.

The aim of this study was to develop eye-drops with cefuroxime (CEF) sodium or vancomycin (VAN) hydrochloride, antibiotics that are instable in water. Anhydrous self-emulsifying oils (SEO) are proposed as a carrier and antibiotics are suspended. In the contact with tear fluid, the formulation should transform into emulsion, with fast dissolution of an antibiotic. CEF or VAN (5% w/w) was suspended in SEO carriers prepared by dissolving surfactants (Tween 20 or Span 80 5% w/w) in Miglyol, castor oil, or olive oil. Formulations with or without sodium citrate (2% w/w) were compared. Six-months or 1-year stability tests were carried out at 40 °C. The content of CEF and VAN was evaluated using HPLC and the potency of the antibiotic was assessed with agar diffusion method. In contact with water, drug particles suspended in SEO dissolved rapidly and o/w emulsion was formed. After 1-year at 40 °C, the content of degradation products was at most 0.5% in CEF and 4.0% in VAN formulations. The agar diffusion assay has shown that CEF and VAN loaded into SEO retained its potency against the sensitive microorganisms comparable to an aqueous solution. Therefore, SEO can be used as a novel carrier for the active substances which may not require improved solubility or absorption but need to be protected from moisture. This is a formulation that can be produced on industrial scale, with no limitation of stability or drug concentration.

Can local application of vancomycin reduce surgical site infection rate after open lumbar fusion surgery?: A multicenter retrospective cohort study.

Surgical site infection (SSI) after posterior open lumbar fusion (POLF) is a major concern for both surgeons and patients. We sought to explore whether local application of vancomycin could decrease the rate of SSI. We reviewed the clinical data of patients who underwent POLF between June 2015 and June 2022 at 3 spinal centers. Patients were divided into those who received local vancomycin (vancomycin group) and those who did not (non-vancomycin group). The SSI rates at 12 months postoperatively were compared between the 2 groups. Although a trend toward a lower infection rate was observed in the vancomycin group than in the non-vancomycin group; the difference was not statistically significant (3.6% vs 5.5%, P = .121). However, we found that the postoperative SSI rate was significantly lower in the vancomycin group than in the non-vancomycin group (4.9% vs 11.4%, P = .041) in patients ≥ 2 fused segments, while there was no significant difference in postoperative SSI rate in patients with single fusion segment (3.1% vs 3.6%, P = .706). The logistic regression analysis indicated that the SSI rate in the non-vancomycin group was approximately 2.498 times higher than that in the vancomycin group (P = .048, odds ratio: 2.498, 95% confidence interval: 1.011-6.617) in patients with ≥2 fused segments. In SSI patients with confirmed pathogens, the SSI rate of Gram-negative bacteria in the vancomycin group was significantly higher than that in the non-vancomycin group (10/14 [71.4%] vs 5/22 [31.8%]), whereas the SSI rate of Gram-positive bacteria in the vancomycin group was significantly lower than that in the non-vancomycin group (4/14 [28.6%] vs 15/22 [68.2%]). Local administration of vancomycin is recommended in patients with ≥2 fused segments as it may facilitate to reduce the postoperative rate of SSI after POLF. Additionally, the local use of vancomycin can decrease the Gram-positive bacterial infections but is not effective against Gram-negative infections, which indirectly leads to an increase in the proportion of Gram-negative infections in SSI patients with confirmed pathogens.

Determination of a vancomycin nephrotoxicity threshold and assessment of target attainment in hematology patients.

An area-under-the-curve (AUC24)-based approach is recommended to guide vancomycin therapeutic drug monitoring (TDM), yet trough concentrations are still commonly used despite associated risks. A definitive toxicity target is lacking, which is important for hematology patients who have a higher risk of nephrotoxicity. The aims were to (1) assess the impact of trough-based TDM on acute kidney injury (AKI) incidence, (2) establish a vancomycin nephrotoxicity threshold, and (3) evaluate the proportion of hematology patients achieving vancomycin therapeutic targets. Retrospective data was collected from 100 adult patients with a hematological malignancy or aplastic anemia who received vancomycin between April 2020 and January 2021. AKI occurrence was determined based on serum creatinine concentrations, and individual pharmacokinetic parameters were estimated using a Bayesian approach. Receiver operating characteristic (ROC) curve analysis was performed to assess the ability of pharmacokinetic indices to predict AKI occurrence. The proportion of patients who achieved target vancomycin exposure was evaluated based on an AUC24/MIC ≥400 and the determined toxicity threshold. The incidence of AKI was 37%. ROC curve analysis indicated a maximum AUC24 of 644 mg.h/L over the treatment period was an important predictor of AKI. By Day 4 of treatment, 29% of treatment courses had supratherapeutic vancomycin exposure, with only 62% of courses achieving AUC24 targets. The identified toxicity threshold supports an AUC24 target range of 400-650 mg.h/L, assuming an MIC of 1 mg/L, to optimize vancomycin efficacy and minimize toxicity. This study highlights high rates of AKI in this population and emphasizes the importance of transitioning from trough-based TDM to an AUC-based approach to improve clinical outcomes.

An induced mutation of ABC-transporter component VraF(K84E) contributes to vancomycin resistance and virulence in Staphylococcus aureus strain MW2.

Staphylococcus aureus is a notorious pathogen responsible for various severe diseases. Due to the emergence of drug-resistant strains, the prevention and treatment of S. aureus infections have become increasingly challenging. Vancomycin is considered to be one of the last-resort drugs for treating most methicillin-resistant S. aureus (MRSA), so it is of great significance to further reveal the mechanism of vancomycin resistance. VraFG is one of the few important ABC (ATP-binding cassette) transporters in S. aureus that can form TCS (two-component systems)/ABC transporter modules. ABC transporters can couple the energy released from ATP hydrolysis to translocate solutes across the cell membrane. In this study, we obtained a strain with decreased vancomycin susceptibility after serial passaging and selection. Subsequently, whole-genome sequencing was performed on this laboratory-derived strain MWA2 and a novel single point mutation was discovered in vraF gene, leading to decreased sensitivity to vancomycin and daptomycin. Furthermore, the mutation reduces autolysis of S. aureus and downregulates the expression of lytM, isaA, and atlA. Additionally, we observed that the mutant has a less net negative surface charge than wild-type strain. We also noted an increase in the expression of the dlt operon and mprF gene, which are associated with cell surface charge and serve to hinder the binding of cationic peptides by promoting electrostatic repulsion. Moreover, this mutation has been shown to enhance hemolytic activity, expand subcutaneous abscesses, reflecting an increased virulence. This study confirms the impact of a point mutation of VraF on S. aureus antibiotic resistance and virulence, contributing to a broader understanding of ABC transporter function and providing new targets for treating S. aureus infections.

Effect of gallium nitrate on the antibacterial activity of vancomycin in methicillin-sensitive and resistant Staphylococcus aureus.

The extension of multidrug-resistant strains of Staphylococcus aureus (S. aureus) is one of the main health challenges in the world, which requires serious solutions to deal with it. Combination therapies using conventional antibiotics and new antibacterial compounds that target different bacterial pathways are effective methods against resistant bacterial infections. Gallium is an iron-like metal that competes with iron for uptake into bacteria and has the potential to disrupt iron-dependent vital processes in bacteria. In this study, we explored the antibacterial effects of gallium nitrate (Ga(NO3)3) and vancomycin alone and in combination with each other on methicillin-sensitive S. aureus (MSSA) and methicillin-resistant S. aureus (MRSA) using microdilution assay and checkerboard test, respectively. Then, their effect on the formation and destruction of biofilms was investigated. Finally, the amount of ROS production in the presence of these two compounds in bacteria was evaluated. The results indicated that the vancomycin/ Ga(NO3)3 combination reduced the MIC of vancomycin in the MRSA strain and had an additive effect on it. Vancomycin plus Ga(NO3)3 reduced the formation of biofilms and increased the destruction of biofilms formed in both strains, especially in the MRSA strain. ROS production was also higher in the combination of vancomycin with Ga(NO3)3 compared to vancomycin alone, especially in MRSA. Therefore, our results showed that Ga(NO3)3 enhances the antibacterial activity of vancomycin and this combination therapy can be considered as a new strategy for the treatment of MRSA infections.

Usefulness of vancomycin treatment individualization via Bayesian algorithms: a 5-year study in critical patients.

Aim: Compare two vancomycin dosing strategies in critical patients with methicillin-resistant Staphylococcus aureus (MRSA) infections, considering the heterogeneity of the dosing regimens administered and their implications for toxicity and efficacy. Materials & methods: Longitudinal retrospective observational study in two patient cohorts (standard dosing vs dosing via Bayesian algorithms). Results: The group of Bayesian algorithms received substantially higher and significantly heterogeneous doses, with an absence of nephrotoxicity. The speed of decrease observed in CRP and PCT was greater for the Bayesian strategy (p = 0.045 and 0.0009, respectively). Conclusion: Applying Bayesian algorithms to vancomycin dosage individualization allows for administering much higher doses than with standard regimens, facilitating a quicker clinical response in the absence of nephrotoxicity.

[Box: see text].

Potent and specific antibiotic combination therapy against Clostridioides difficile.

Keratinicyclins and keratinimicins are recently discovered glycopeptide antibiotics. Keratinimicins show broad-spectrum activity against Gram-positive bacteria, while keratinicyclins form a new chemotype by virtue of an unusual oxazolidinone moiety and exhibit specific antibiosis against Clostridioides difficile. Here we report the mechanism of action of keratinicyclin B (KCB). We find that steric constraints preclude KCB from binding peptidoglycan termini. Instead, KCB inhibits C. difficile growth by binding wall teichoic acids (WTAs) and interfering with cell wall remodeling. A computational model, guided by biochemical studies, provides an image of the interaction of KCB with C. difficile WTAs and shows that the same H-bonding framework used by glycopeptide antibiotics to bind peptidoglycan termini is used by KCB for interacting with WTAs. Analysis of KCB in combination with vancomycin (VAN) shows highly synergistic and specific antimicrobial activity, and that nanomolar combinations of the two drugs are sufficient for complete growth inhibition of C. difficile, while leaving common commensal strains unaffected.

Physicochemical and Biological Properties of Vancomycin-Containing Antibacterial Polysaccharide Gels for Biocomposite Bone Implant Impregnation.

Polymeric drugs containing up to 60% by weight of the antibiotic vancomycin were synthesized based on dextran carriers activated with epichlorohydrin. Vancomycin was covalently bound, involving the primary amino group of the molecule through the hydroxypropyl radical to the C6 position of the anhydroglucose units of the dextran main chain. Covalent binding is necessary to prevent spontaneous release of the antibiotic from the gel, thereby reducing the risk of bacterial multiresistance. Antibacterial depot gels were obtained from those polymers, containing up to 17.5% by weight of polysaccharide with a cross-linking density of q = 3-5 nodes per macromolecule for the deposition of another type of drugs not covalently bound to the polymer gel. They were used to coat the surface of the internal pores of biocomposite bone implants based on bovine cancellous bone used in orthopedics. The chemical structure of the polymer was studied using 13C NMR spectroscopy and matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry. The stiffness of the gels was evaluated by the values of the accumulation modulus G' = 170-270 kPa and the loss modulus G″ = 3.7-4.2 kPa determined on a rheometer. Their values are close to those typical for materials used to replace soft tissue in plastic surgery. The minimum inhibitory concentration of the gels against Staphylococcus aureus P209 depends on the antibiotic content in the polymer. It equals 2.5 mg/L for vancomycin we used and 100 mg/L for a polymer containing 50% by weight of covalently bound antibiotic. The cytotoxic concentration measured with cell culture HEK 293T exceeds 1200 mg/L in 24 h exposure. The release dynamics of drugs not covalently bound to dextran from the depot gel were studied using fluorescein as a model. The release time is independent of the gel density and lasts up to 6 days for a 2 mm thick layer. Both the gel and the bone implants impregnated with it maintained consistently high antibacterial activity throughout the experiment, up to its completion after 168 h, with the local concentration of the released antibiotic at the site of bacterial attack exceeding the therapeutic level by 200 times.

Lytic transglycosylase repertoire diversity enables intrinsic antibiotic resistance and daughter cell separation in Escherichia coli under acidic stress.

Peptidoglycan (PG) is an important architectural element that imparts physical toughness and rigidity to the bacterial envelope. It is also a dynamic structure that undergoes continuous turnover or autolysis. Escherichia coli possesses redundant PG degradation enzymes responsible for PG turnover; however, the advantage afforded by the existence of numerous PG degradation enzymes remains incompletely understood. In this study, we elucidated the physiological roles of MltE and MltC, members of the lytic transglycosylase (LTG) family that catalyze the cleavage of glycosidic bonds between disaccharide subunits within PG strands. MltE and MltC are acidic LTGs that exhibit increased enzymatic activity and protein levels under acidic pH conditions, respectively, and deletion of these two LTGs results in a pronounced growth defect at acidic pH. Furthermore, inactivation of these two LTGs induces increased susceptibility at acidic pH against various antibiotics, particularly vancomycin, which seems to be partially caused by elevated membrane permeability. Intriguingly, inactivation of these LTGs induces a chaining morphology, indicative of daughter cell separation defects, only under acidic pH conditions. Simultaneous deletion of PG amidases, known contributors to daughter cell separation, exacerbates the chaining phenotype at acidic pH. This suggests that the two LTGs may participate in the cleavage of glycan strands between daughter cells under acidic pH conditions. Collectively, our findings highlight the role of LTG repertoire diversity in facilitating bacterial survival and antibiotic resistance under stressful conditions.

The zebrafish embryo model: unveiling its potential for investigating phage therapy against methicillin-resistant Staphylococcus aureus infection.

Staphylococcus aureus is a pathogenic bacterium responsible for a broad spectrum of infections, including cutaneous, respiratory, osteoarticular, and systemic infections. It poses a significant clinical challenge due to its ability to develop antibiotic resistance. This resistance limits therapeutic options, increases the risk of severe complications, and underscores the urgent need for new strategies to address this threat, including the investigation of treatments complementary to antibiotics. The evaluation of novel antimicrobial agents often employs animal models, with the zebrafish embryo model being particularly interesting for studying host-pathogen interactions, establishing itself as a crucial tool in this field. For the first time, this study presents a zebrafish embryo model for the in vivo assessment of bacteriophage efficacy against S. aureus infection. A localized infection was induced by microinjecting either methicillin-resistant S. aureus (MRSA) or methicillin-susceptible S. aureus (MSSA). Subsequent treatments involved administering either bacteriophage, vancomycin (the reference antibiotic for MRSA), or a combination of both via the same route to explore potential synergistic effects. Our findings indicate that the bacteriophage was as effective as vancomycin in enhancing survival rates, whether used alone or in combination. Moreover, bacteriophage treatment appears to be even more effective in reducing the bacterial load in S. aureus-infected embryos post-treatment than the antibiotic. Our study validates the use of the zebrafish embryo model and highlights its potential as a valuable tool in assessing bacteriophage efficacy treatments in vivo.