RNA polymerase stalling-derived genome instability underlies ribosomal antibiotic efficacy and resistance evolution.

Bacteria often evolve antibiotic resistance through mutagenesis. However, the processes causing the mutagenesis have not been fully resolved. Here, we find that a broad range of ribosome-targeting antibiotics cause mutations through an underexplored pathway. Focusing on the clinically important aminoglycoside gentamicin, we find that the translation inhibitor causes genome-wide premature stalling of RNA polymerase (RNAP) in a loci-dependent manner. Further analysis shows that the stalling is caused by the disruption of transcription-translation coupling. Anti-intuitively, the stalled RNAPs subsequently induce lesions to the DNA via transcription-coupled repair. While most of the bacteria are killed by genotoxicity, a small subpopulation acquires mutations via SOS-induced mutagenesis. Given that these processes are triggered shortly after antibiotic addition, resistance rapidly emerges in the population. Our work reveals a mechanism of action of ribosomal antibiotics, illustrates the importance of dissecting the complex interplay between multiple molecular processes in understanding antibiotic efficacy, and suggests new strategies for countering the development of resistance.

Silver nanoparticle with potential antimicrobial and antibiofilm efficiency against multiple drug resistant, extensive drug resistant Pseudomonas aeruginosa clinical isolates.

BACKGROUND: The study aims to investigate the effect of combining silver nanoparticles (AGNPs) with different antibiotics on multi-drug resistant (MDR) and extensively drug resistant (XDR) isolates of Pseudomonas aeruginosa (P. aeruginosa) and to investigate the mechanism of action of AGNPs. METHODS: AGNPs were prepared by reduction of silver nitrate using trisodium citrate and were characterized by transmission electron microscope (TEM) in addition to an assessment of cytotoxicity. Clinical isolates of P. aeruginosa were collected, and antimicrobial susceptibility was conducted. Multiple Antibiotic Resistance (MAR) index was calculated, and bacteria were categorized as MDR or XDR. Minimum inhibitory concentration (MIC) of gentamicin, ciprofloxacin, ceftazidime, and AGNPs were determined. The mechanism of action of AGNPs was researched by evaluating their effect on biofilm formation, swarming motility, protease, gelatinase, and pyocyanin production. Real-time PCR was performed to investigate the effect on the expression of genes encoding various virulence factors. RESULTS: TEM revealed the spherical shape of AGNPs with an average particle size of 10.84 ± 4.64 nm. AGNPS were safe, as indicated by IC50 (42.5 µg /ml). The greatest incidence of resistance was shown against ciprofloxacin which accounted for 43% of the bacterial isolates. Heterogonous resistance patterns were shown in 63 isolates out of the tested 107. The MAR indices ranged from 0.077 to 0.84. Out of 63 P. aeruginosa isolates, 12 and 13 were MDR and XDR, respectively. The MIC values of AGNPs ranged from 2.65 to 21.25 µg /ml. Combination of AGNPs with antibiotics reduced their MIC by 5-9, 2-9, and 3-10Fold in the case of gentamicin, ceftazidime, and ciprofloxacin, respectively, with synergism being evident. AGNPs produced significant inhibition of biofilm formation and decreased swarming motility, protease, gelatinase and pyocyanin production. PCR confirmed the finding, as shown by decreased expression of genes encoding various virulence factors. CONCLUSION: AGNPs augment gentamicin, ceftazidime, and ciprofloxacin against MDR and XDR Pseudomonas isolates. The efficacy of AGNPs can be attributed to their effect on the virulence factors of P. aeruginosa. The combination of AGNPs with antibiotics is a promising strategy to attack resistant isolates of P. aeruginosa.

Optimized Electrophoretic Deposition of Chitosan/Mesoporous Glass Nanoparticles with Gentamicin on Titanium Implants: Enhancing Hemocompatibility and Antibacterial Activity.

Titanium-based implants have long been studied and used for applications in bone tissue engineering, thanks to their outstanding mechanical properties and appropriate biocompatibility. However, many implants struggle with osseointegration and attachment and can be vulnerable to the development of infections. In this work, we have developed a composite coating via electrophoretic deposition, which is both bioactive and antibacterial. Mesoporous bioactive glass particles with gentamicin were electrophoretically deposited onto a titanium substrate. In order to validate the hypothesis that the quantity of particles in the coatings is sufficiently high and uniform in each deposition process, an easy-to-use image processing algorithm was designed to minimize human dependence and ensure reproducible results. The addition of loaded mesoporous particles did not affect the good adhesion of the coating to the substrate although roughness was clearly enhanced. After 7 days of immersion, the composite coatings were almost dissolved and released, but phosphate-related compounds started to nucleate at the surface. With a simple and low-cost technique like electrophoretic deposition, and optimized stir and suspension times, we were able to synthesize a hemocompatible coating that significantly improves the antibacterial activity when compared to the bare substrate for both Gram-positive and Gram-negative bacteria.

Synergistic antibacterial activity of ceftazidime-avibactam in combination with colistin, gentamicin, amikacin, and fosfomycin against carbapenem-resistant Klebsiella pneumoniae.

Carbapenem-resistant Klebsiella pneumoniae (CPKP) infections seriously threaten global public health. The main objective of this study was to assess the in-vitro synergistic activity of ceftazidime-avibactam (CZA) in combination with colistin (COL), amikacin (AK), gentamicin (GEN), and fosfomycin (FOS) against CPKP isolates. The secondary goal was to determine the antibiotic susceptibility performance of BD Phoenix. OXA-48 (49.1%) was the predominant carbapenemase, followed by KPC (29.1%). We used the broth microdilution (BMD) method to determine the minimum inhibitory concentrations (MICs) of CZA, COL, AK, and GEN. Meanwhile, the MICs of FOS were determined by the agar dilution (AD) method. To examine the antibacterial activity of CZA, we conducted a checkerboard assay (CBA) with COL, AK, GEN, and FOS against CRKP isolates. We randomly selected three strains and performed synergy testing via time-kill assay (TKA). CRKP isolates were 89.1% susceptible to CZA, 16.4% to COL, 21.8% to GEN, and 29.1% to AK using BMD, 47.3% to FOS by AD. The most synergistic effects were observed in the combination of CZA-COL (78.2%) and CZA-FOS (63.6%). Given the limited therapeutic options for treating severe CRKP infections, combining CZA with COL and FOS may enhance in-vitro activity against clinical CRKP isolates.

Regulation of the p53/SLC7A11/GPX4 Pathway by Gentamicin Induces Ferroptosis in HEI-OC1 Cells.

BACKGROUND: Gentamicin is a commonly used aminoglycoside antibiotic, with ototoxicity as a significant side effect. Ferroptosis, an iron-dependent form of cell death, has been implicated in a variety of disorders. Whether ferroptosis impacts gentamicin ototoxicity is not yet known. The current work used an in-vitro model to examine the influence of gentamicin-induced ferroptosis on cochlear hair cell damage and probable molecular biological pathways. METHODS: House Ear Institute-Organ of Corti 1 (HEI-OC1) cells were treated with different concentrations of gentamicin for 24 hours, with or without ferrostatin-1 pretreatment, to observe gentamicin-induced ferroptosis. The role of p53/solute carrier family 7 member 11 (SLC7A11)/glutathione peroxidase 4 (GPX4) signaling in gentamicin-induced ferroptosis was explored by pretreating cells with the p53 inhibitor pifithrin-α (PFT-α). We investigated the effect of gentamicin on cells by assessing cell viability. Cellular proteins were isolated and Western blots were performed to detect changes in the expression of p53, SLC7A11, and GPX4. Fluorescence staining was used to assess levels of reactive oxygen species. An enzymatic detection kit was used to detect glutathione, Fe, and malondialdehyde markers. RESULTS: Gentamicin reduced cell viability, glutathione content, and SLC7A11 and GPX4 protein levels, and increased levels of p53 protein, reactive oxygen species, malondialdehyde, and Fe. These effects were largely blocked by pretreatment with ferrostatin-1. Pretreatment with the p53 inhibitor PFT-α prevented the gentamicin-induced reduction in SLC7A11 and GPX4, which alleviated several features of ferroptosis including glutathione depletion, iron overload, and lipid peroxidation build-up. CONCLUSION: Gentamicin induces ferroptosis in the HEI-OC1 cell line, and the mechanism may be related to the p53/SLC7A11/GPX4 signaling pathway.

In vitro identification of underutilized ß-lactam combinations against methicillin-resistant Staphylococcus aureus bacteremia isolates.

Methicillin-resistant Staphylococcus aureus (MRSA) bacteremia is a serious clinical challenge with high mortality rates. Antibiotic combination therapy is currently used in cases of persistent infection; however, the limited development of new antibiotics will likely increase the need for combination therapy, and better methods are needed for identifying effective combinations for treating persistent bacteremia. To identify pairwise combinations with the most consistent potential for benefit compared to monotherapy with a primary anti-MRSA agent, we conducted a systematic study with an in vitro high-throughput methodology. We tested daptomycin and vancomycin each in combination with gentamicin, rifampicin, cefazolin, and oxacillin, and ceftaroline with daptomycin, gentamicin, and rifampicin. Combining cefazolin with daptomycin lowered the daptomycin concentration required to reach 95% growth inhibition (IC95) for all isolates tested and lowered daptomycin IC95 below the sensitivity breakpoint for five out of six isolates that had daptomycin minimum inhibitory concentrations at or above the sensitivity breakpoint. Similarly, vancomycin IC95s were decreased when vancomycin was combined with cefazolin for 86.7% of the isolates tested. This was a higher percentage than was achieved by adding any other secondary antibiotic to vancomycin. Adding rifampicin to daptomycin or vancomycin did not always reduce IC95s and failed to produce synergistic interaction in any of the isolates tested; the addition of rifampicin to ceftaroline was frequently synergistic and always lowered the amount of ceftaroline required to reach the IC95. These analyses rationalize further in vivo evaluation of three drug pairs for MRSA bacteremia: daptomycin+cefazolin, vancomycin+cefazolin, and ceftaroline+rifampicin.IMPORTANCEBloodstream infections caused by methicillin-resistant Staphylococcus aureus (MRSA) have a high mortality rate despite the availability of vancomycin, daptomycin, and newer antibiotics including ceftaroline. With the slow output of the antibiotic pipeline and the serious clinical challenge posed by persistent MRSA infections, better strategies for utilizing combination therapy are becoming increasingly necessary. We demonstrated the value of a systematic high-throughput approach, adapted from prior work testing antibiotic combinations against tuberculosis and other mycobacteria, by using this approach to test antibiotic pairs against a panel of MRSA isolates with diverse patterns of antibiotic susceptibility. We identified three antibiotic pairs-daptomycin+cefazolin, vancomycin+cefazolin, and ceftaroline+rifampicin-where the addition of the second antibiotic improved the potency of the first antibiotic across all or most isolates tested. Our results indicate that these pairs warrant further evaluation in the clinical setting.

Antimicrobial peptides and proteins as alternative antibiotics for porcine semen preservation.

BACKGROUND: Antimicrobial resistance (AMR) is nowadays a major emerging challenge for public health worldwide. The over- and misuse of antibiotics, including those for cell culture, are promoting AMR while also encouraging the research and employment of alternative drugs. The addition of antibiotics to the cell media is strongly recommended in sperm preservation, being gentamicin the most used for boar semen. Because of its continued use, several bacterial strains present in boar semen have developed resistance to this antibiotic. Antimicrobial peptides and proteins (AMPPs) are promising candidates as alternative antibiotics because their mechanism of action is less likely to promote AMR. In the present study, we tested two AMPPs (lysozyme and nisin; 50 and 500 µg/mL) as possible substitutes of gentamicin for boar semen preservation up to 48 h of storage. RESULTS: We found that both AMPPs improved sperm plasma membrane and acrosome integrity during semen storage. The highest concentration tested for lysozyme also kept the remaining sperm parameters unaltered, at 48 h of semen storage, and reduced the bacterial load at comparable levels of the samples supplemented with gentamicin (p > 0.05). On the other hand, while nisin (500 µg/mL) reduced the total Enterobacteriaceae counts, it also decreased the rapid and progressive sperm population and the seminal oxidation-reduction potential (p < 0.05). CONCLUSIONS: The protective effect of lysozyme on sperm function together with its antimicrobial activity and inborn presence in body fluids, including semen and cervical mucus, makes this enzyme a promising antimicrobial agent for boar semen preservation.

Upregulated Palmitoleate and Oleate Production in Escherichia coli Promotes Gentamicin Resistance.

Metabolic reprogramming mediates antibiotic efficacy. However, metabolic adaptation of microbes evolving from antibiotic sensitivity to resistance remains undefined. Therefore, untargeted metabolomics was conducted to unveil relevant metabolic reprogramming and potential intervention targets involved in gentamicin resistance. In total, 61 metabolites and 52 metabolic pathways were significantly altered in gentamicin-resistant E. coli. Notably, the metabolic reprogramming was characterized by decreases in most metabolites involved in carbohydrate and amino acid metabolism, and accumulation of building blocks for nucleotide synthesis in gentamicin-resistant E. coli. Meanwhile, fatty acid metabolism and glycerolipid metabolism were also significantly altered in gentamicin-resistant E. coli. Additionally, glycerol, glycerol-3-phosphate, palmitoleate, and oleate were separately defined as the potential biomarkers for identifying gentamicin resistance in E. coli. Moreover, palmitoleate and oleate could attenuate or even abolished killing effects of gentamicin on E. coli, and separately increased the minimum inhibitory concentration of gentamicin against E. coli by 2 and 4 times. Furthermore, palmitoleate and oleate separately decreased intracellular gentamicin contents, and abolished gentamicin-induced accumulation of reactive oxygen species, indicating involvement of gentamicin metabolism and redox homeostasis in palmitoleate/oleate-promoted gentamicin resistance in E. coli. This study identifies the metabolic reprogramming, potential biomarkers and intervention targets related to gentamicin resistance in bacteria.

The Effect of Gentamicin on the Motility of Hormonally Induced Spermatozoa of Toad Bufo bufo during Storage at 4°C.

We studied the effect of antibiotic gentamicin at concentrations of 0.05, 0.1, 0.2, 0.4, and 1 mg/ml on the maintenance of sperm motility of the common toad Bufo bufo during cold storage of spermic urine samples at 4°C. Parameters of sperm motility during storage of samples with gentamicin at concentrations of 0.05-0.4 mg/ml did not differ significantly, but were higher (p<0.0001) than in the control (storage without antibiotic). Gentamicin at a concentration of 1 mg/ml had a negative effect on sperm motility. After 2 weeks of storage of toad spermic urine samples with gentamicin, the largest number of sperm was preserved when using antibiotic at a concentration of 0.4 mg/ml.

Clinical and Microbiological Characteristics of Bacteremia Caused by Carbapenemase-producing Enterobacterales in Minami Ibaraki Area, Japan.

Although recent propagation of carbapenemase-producing Enterobacterales (CPE) has become a problem worldwide, the picture of CPE infection in Japan has not fully been elucidated. In this study, we examined clinical and microbiological characteristics of invasive CPE infection occurring at 8 hospitals in Minami Ibaraki Area between July 2001 to June 2017. Of 7294 Enterobacterales strains isolated from independent cases of bacteremia and/or meningitis, 10 (0.14%) were CPE (8 Enterobacter cloacae-complex, 1 Escherichia coli, and 1 Edwardsiella tarda）, all of which had the blaIMP-1 gene and susceptible to gentamicin and trimethoprim/sulfamethoxazole. These strains were isolated from 7 adult and 2 infant bacteremia (1 infant patient developed CPE bacteremia twice) after 2007. The most common portal of entry was intravenous catheters. All of the adult patients were recovered, while the infant patients eventually died. Genomic analyses showed that the 8 E. cloacae-complex strains were classified into 5 groups, each of which was exclusively detected in specific facilities at intervals of up to 3 years, suggesting persistent colonization in the facilities. This study showed that invasive CPE infection in the area was rare, caused by IMP-1-type CPE having susceptibility to various antibiotics, and nonfatal among adult patients.

Reversal of gentamicin sulfate resistance in avian pathogenic Escherichia coli by matrine combined with berberine hydrochloride.

In recent years, the evolution of antibiotic resistance has led to the inefficacy of several antibiotics, and the reverse of resistance was a novel method to solve this problem. We previously demonstrated that matrine (Mat) and berberine hydrochloride (Ber) had a synergistic effect against multidrug-resistant Escherichia coli (MDREC). This study aimed to demonstrate the effect of Mat combined with Ber in reversing the resistance of MDREC. The MDREC was sequenced passaged in the presence of Mat, Ber, and a combination of Mat and Ber, which did not affect its growth. The reverse rate was up to 39.67% after MDREC exposed to Mat + Ber for 15 days. The strain that reversed resistance was named drug resistance reversed E. coli (DRREC) and its resistance to ampicillin, streptomycin, gentamicin, and tetracycline was reversed. The MIC of Gentamicin Sulfate (GS) against DRREC decreased 128-fold to 0.63 µg/mL, and it was stable within 20 generations. Furthermore, the susceptible phenotype of DRREC remained stable within 20 generations, as well. The LD50 of DRREC for chickens was 8.69 × 109 CFU/mL. qRT-PCR assays revealed that the transcript levels of antibiotic-resistant genes and virulence genes in the DRREC strain were significantly lower than that in the MDREC strain (P < 0.05). In addition, GS decreased the death, decreased the bacterial loading in organs, alleviated the injury of the spleen and liver, and decreased the cytokine levels in the chickens infected by the DRREC strain. In contrast, the therapeutic effect of GS in chickens infected with MDREC was not as evident. These findings suggest that the combination of Mat and Ber has potential for reversing resistance to MDREC.

Quantification of early biofilm growth in microtiter plates through a novel image analysis software.

Given the significant impact of biofilms on human health and material corrosion, research in this field urgently needs more accessible techniques to facilitate the testing of new control agents and general understanding of biofilm biology. Microtiter plates offer a convenient format for standardized evaluations, including high-throughput assays of alternative treatments and molecular modulators. This study introduces a novel Biofilm Analysis Software (BAS) for quantifying biofilms from microtiter plate images. We focused on early biofilm growth stages and compared BAS quantification to common techniques: direct turbidity measurement, intrinsic fluorescence detection linked to pyoverdine production, and standard crystal violet staining which enables image analysis and optical density measurement. We also assessed their sensitivity for detecting subtle growth effects caused by cyclic AMP and gentamicin. Our results show that BAS image analysis is at least as sensitive as the standard method of spectrophotometrically quantifying the crystal violet retained by biofilms. Furthermore, we demonstrated that bacteria adhered after short incubations (from 10 min to 4 h), isolated from planktonic populations by a simple rinse, can be monitored until their growth is detectable by intrinsic fluorescence, BAS analysis, or resolubilized crystal violet. These procedures are widely accessible for many laboratories, including those with limited resources, as they do not require a spectrophotometer or other specialized equipment.

α-Ketoglutarate downregulates thiosulphate metabolism to enhance antibiotic killing.

Potentiation of the effects of currently available antibiotics is urgently required to tackle the rising antibiotics resistance. The pyruvate (P) cycle has been shown to play a critical role in mediating aminoglycoside antibiotic killing, but the mechanism remains unexplored. In this study, we investigated the effects of intermediate metabolites of the P cycle regarding the potentiation of gentamicin. We found that α-ketoglutarate (α-KG) has the best synergy with gentamicin compared to the other metabolites. This synergistic killing effect was more effective with aminoglycosides than other types of antibiotics, and it was effective against various types of bacterial pathogens. Using fish and mouse infection models, we confirmed that the synergistic killing effect occurred in vivo. Furthermore, functional proteomics showed that α-KG downregulated thiosulphate metabolism. Upregulation of thiosulphate metabolism by exogenous thiosulphate counteracted the killing effect of gentamicin. The role of thiosulphate metabolism in antibiotic resistance was further confirmed using thiosulphate reductase knockout mutants. These mutants were more sensitive to gentamicin killing, and less tolerant to antibiotics compared to their parental strain. Thus, our study highlights a strategy for potentiating antibiotic killing by using a metabolite that reduces antibiotic resistance.

Gentamicin loaded niosomes against intracellular uropathogenic Escherichia coli strains.

Urinary tract infections (UTIs) are the most common bacterial infections and uropathogenic Escherichia coli (UPEC) is the main etiological agent of UTIs. UPEC can persist in bladder cells protected by immunological defenses and antibiotics and intracellular behavior leads to difficulty in eradicating the infection. The aim of this paper is to design, prepare and characterize surfactant-based nanocarriers (niosomes) able to entrap antimicrobial drug and potentially to delivery and release antibiotics into UPEC-infected cells. In order to validate the proposed drug delivery system, gentamicin, was chosen as "active model drug" due to its poor cellular penetration. The niosomes physical-chemical characterization was performed combining different techniques: Dynamic Light Scattering Fluorescence Spectroscopy, Transmission Electron Microscopy. Empty and loaded niosomes were characterized in terms of size, ζ-potential, bilayer features and stability. Moreover, Gentamicin entrapped amount was evaluated, and the release study was also carried out. In addition, the effect of empty and loaded niosomes was studied on the invasion ability of UPEC strains in T24 bladder cell monolayers by Gentamicin Protection Assay and Confocal Microscopy. The observed decrease in UPEC invasion rate leads us to hypothesize a release of antibiotic from niosomes inside the cells. The optimization of the proposed drug delivery system could represent a promising strategy to significatively enhance the internalization of antimicrobial drugs.

Human-Derived collagen hydrogel as an antibiotic vehicle for topical treatment of bacterial biofilms.

The complexity of chronic wounds creates difficulty in effective treatments, leading to prolonged care and significant morbidity. Additionally, these wounds are incredibly prone to bacterial biofilm development, further complicating treatment. The current standard treatment of colonized superficial wounds, debridement with intermittent systemic antibiotics, can lead to systemic side-effects and often fails to directly target the bacterial biofilm. Furthermore, standard of care dressings do not directly provide adequate antimicrobial properties. This study aims to assess the capacity of human-derived collagen hydrogel to provide sustained antibiotic release to disrupt bacterial biofilms and decrease bacterial load while maintaining host cell viability and scaffold integrity. Human collagen harvested from flexor tendons underwent processing to yield a gellable liquid, and subsequently was combined with varying concentrations of gentamicin (50-500 mg/L) or clindamycin (10-100 mg/L). The elution kinetics of antibiotics from the hydrogel were analyzed using liquid chromatography-mass spectrometry. The gel was used to topically treat Methicillin-resistant Staphylococcus aureus (MRSA) and Clostridium perfringens in established Kirby-Bauer and Crystal Violet models to assess the efficacy of bacterial inhibition. 2D mammalian cell monolayers were topically treated, and cell death was quantified to assess cytotoxicity. Bacteria-enhanced in vitro scratch assays were treated with antibiotic-embedded hydrogel and imaged over time to assess cell death and mobility. Collagen hydrogel embedded with antibiotics (cHG+abx) demonstrated sustained antibiotic release for up to 48 hours with successful inhibition of both MRSA and C. perfringens biofilms, while remaining bioactive up to 72 hours. Administration of cHG+abx with antibiotic concentrations up to 100X minimum inhibitory concentration was found to be non-toxic and facilitated mammalian cell migration in an in vitro scratch model. Collagen hydrogel is a promising pharmaceutical delivery vehicle that allows for safe, precise bacterial targeting for effective bacterial inhibition in a pro-regenerative scaffold.

In vitro effects of alginate lyase SG4 + produced by Paenibacillus lautus alone and combined with antibiotics on biofilm formation by mucoid Pseudomonas aeruginosa.

Alginate is a major extra polymeric substance in the biofilm formed by mucoid Pseudomonas aeruginosa. It is the main proven perpetrator of lung infections in patients suffering from cystic fibrosis. Alginate lyases are very important in the treatment of cystic fibrosis. This study evaluated the role of standalone and in conjugation, effect of alginate lyase of SG4 + isolated from Paenibacillus lautus in enhancing in vitro bactericidal activity of gentamicin and amikacin on mucoid P. aeruginosa. Using Response Surface Methodology (RSM) alginate lyase SG4 + production was optimized in shake flask and there 8.49-fold enhancement in enzyme production. In fermenter, maximum growth (10.15 mg/ml) and alginate lyase (1.46 International Units) production, 1.71-fold was increased using Central Composite Design (CCD). Further, fermentation time was reduced from 48 to 20 h. To the best of our knowledge this is the first report in which CCD was used for fermenter studies to optimize alginate lyase production. The Km and Vmax of purified enzyme were found to be 2.7 mg/ml and 0.84 mol/ml-min, respectively. The half-life (t 1/2) of purified alginate lyase SG4 + at 37 °C was 180 min. Alginate lyase SG4 + in combination with gentamicin and amikacin eradiated 48.4- 52.3% and 58- 64.6%, alginate biofilm formed by P. aeruginosa strains, respectively. The study proves that alginate lyase SG4 + has excellent exopolysaccharide disintegrating ability and may be useful in development of potent therapeutic agent to treat P. aeruginosa biofilms.

Increased tolerance to commonly used antibiotics in a Pseudomonas aeruginosa ex vivo porcine keratitis model.

Introduction. Bacterial keratitis, particularly caused by Pseudomonas aeruginosa, is challenging to treat because of multi-drug tolerance, often associated with the formation of biofilms. Antibiotics in development are typically evaluated against planktonic bacteria in a culture medium, which may not accurately represent the complexity of infections in vivo.Hypothesis/Gap Statement. Developing a reliable, economic ex vivo keratitis model that replicates some complexity of tissue infections could facilitate a deeper understanding of antibiotic efficacy, thus aiding in the optimization of treatment strategies for bacterial keratitis.Methodology. Here we investigated the efficacy of three commonly used antibiotics (gentamicin, ciprofloxacin and meropenem) against Pseudomonas aeruginosa cytotoxic strain PA14 and invasive strain PA01 using an ex vivo porcine keratitis model.Results. Both strains of P. aeruginosa were susceptible to the MIC of the three tested antibiotics. However, significantly higher concentrations were necessary to inhibit bacterial growth in the minimum biofilm eradication concentration (MBEC) assay, with both strains tolerating concentrations greater than 512 mg l-1 of meropenem. When MIC and higher concentrations than MBEC (1024 mg l-1) of antibiotics were applied, ciprofloxacin exhibited the highest potency against both P. aeruginosa strains, followed by meropenem, while gentamicin showed the least potency. Despite this, none of the antibiotic concentrations used effectively cleared the infection, even after 18 h of continuous exposure.Conclusions. Further exploration of antibiotic concentrations and aligning dosing with clinical studies to validate the model is needed. Nonetheless, our ex vivo porcine keratitis model could be a valuable tool for assessing antibiotic efficacy.

Triton X-100 counteracts antibiotic resistance of Enterococcus faecalis: An in vitro study.

OBJECTIVES: The high prevalence of antibiotic-resistant bacteria poses a threat to the global public health. The appropriate use of adjuvants to restore the antimicrobial activity of antibiotics against resistant bacteria could be an effective strategy for combating antibiotic resistance. In this study, we investigated the counteraction of Triton X-100 (TX-100) and the mechanisms underlying the antibiotic resistance of Enterococcus faecalis (E. faecalis). METHODS: Standard, wild-type (WT), and induced antibiotic-resistant E. faecalis strains were used in this study. In vitro antibacterial experiments were conducted to evaluate the antimicrobial activities of gentamicin sulfate and ciprofloxacin hydrochloride in the presence and absence of 0.02 % TX-100 against both planktonic and biofilm bacteria. Transcriptomic and untargeted metabolomic analyses were performed to explore the molecular mechanisms of TX-100 as an antibiotic adjuvant. Additionally, membrane permeability, membrane potential, glycolysis-related enzyme activity, intracellular adenosine triphosphate (ATP), and expression levels of virulence genes were assessed. The biocompatibility of different drug combinations was also evaluated. RESULTS: A substantially low TX-100 concentration improved the antimicrobial effects of gentamicin sulfate or ciprofloxacin hydrochloride against antibiotic-resistant E. faecalis. Mechanistic studies demonstrated that TX-100 increased cell membrane permeability and dissipated membrane potential. Moreover, antibiotic resistance and pathogenicity of E. faecalis were attenuated by TX-100 via downregulation of the ABC transporter, phosphotransferase system (PTS), and ATP supply. CONCLUSIONS: TX-100 enhanced the antimicrobial activity of gentamicin sulfate and ciprofloxacin hydrochloride at a low concentration by improving antibiotic susceptibility and attenuating antibiotic resistance and pathogenicity of E. faecalis. CLINICAL SIGNIFICANCE: These findings provide a theoretical basis for developing new root canal disinfectants that can reduce antibiotic resistance.

Hierarchical antibiotic delivery system based on calcium phosphate cement/montmorillonite-gentamicin sulfate with drug release pathways.

Antibiotic-loaded calcium phosphate cement (CPC) has emerged as a promising biomaterial for drug delivery in orthopedics. However, there are problems such as the burst release of antibiotics, low cumulative release ratio, inappropriate release cycle, inferior mechanical strength, and poor anti-collapse properties. In this research, montmorillonite-gentamicin (MMT-GS) was fabricated by solution intercalation method and served as the drug release pathways in CPC to avoid burst release of GS, achieving promoted cumulative release ratios and a release cycle matched the time of inflammatory response. The results indicated that the highest cumulative release ratio and release concentration of GS in CPC/MMT-GS was 94.1 ± 2.8 % and 1183.05 µg/mL, and the release cycle was up to 504 h. In addition, the hierarchical GS delivery system was divided into three stages, and the kinetics followed the Korsmeyer-Peppas model, the zero-order model, and the diffusion-dissolution model, respectively. Meanwhile, the compressive strength of CPC/MMT-GS was up to 51.33 ± 3.62 MPa. Antibacterial results demonstrated that CPC/MMT-GS exhibited excellent in vitro long-lasting antibacterial properties to E. coli and S. aureus. Furthermore, CPC/MMT-GS promoted osteoblast proliferation and exhibited excellent in vivo histocompatibility. Therefore, CPC/MMT-GS has favorable application prospects in the treatment of bone defects with bacterial infections and inflammatory reactions.

A comprehensive study on Geranium robertianum L. antibacterial potential.

AIMS: The research aimed to optimize the ultrasound-assisted extraction of secondary metabolites and the antibacterial activity of the plant species Geranium robertianum. The phytochemical profiles of the optimized extracts, as well as their antibacterial and synergistic activity with an antibiotic and their potential mechanisms of action and cytotoxicity, were examined. METHODS AND RESULTS: Response Surface Methodology was used to optimize extraction conditions. Optimized ethanol and acetone extracts were tested via microdilution, checkerboard, time-kill kinetics, and cell membrane permeability methods. The extracts displayed broad antibacterial activity with minimum inhibitory concentrations ranging from 1.25 to 20 mg ml-1. In addition, the extract synergistically reacted with gentamicin against gentamicin-resistant strains of Escherichia coli and Staphylococcus aureus, enhancing the efficacy of the antibiotic up to 32-fold. The extracts demonstrated strain-dependent bactericidal activity in a 24-h time interval. They increase the permeability of the cell membrane, thus disrupting its normal functioning. The cytotoxic concentration (CC50) on human keratinocytes was 1771.24 ± 5.78 µg ml-1 for ethanol extract, and 958.01 ± 6.14 µg ml-1 for acetone extract. Kaempferol, ellagic acid, quercetin, and rutin were recognized as the main components in both extracts. CONCLUSIONS: The findings of this study indicate that the extracts of G. robertianum can be considered as potential natural antibacterial agents in the control of microorganisms.