Eradication of Staphylococcus aureus in Implant-Associated Osteomyelitis by an Injectable In Situ-Forming Depot Antibiotics Delivery System.

BACKGROUND: Bone infections with Staphylococcus aureus are notoriously difficult to treat and have high recurrence rates. Local antibiotic delivery systems hold the potential to achieve high in situ antibiotic concentrations, which are otherwise challenging to achieve via systemic administration. Existing solutions have been shown to confer suboptimal drug release and distribution. Here we present and evaluate an injectable in situ-forming depot system termed CarboCell. The CarboCell technology provides sustained and tuneable release of local high-dose antibiotics. METHODS: CarboCell formulations of levofloxacin or clindamycin with or without antimicrobial adjuvants cis-2-decenoic acid or cis-11-methyl-2-dodecenoic acid were tested in experimental rodent and porcine implant-associated osteomyelitis models. In the porcine models, debridement and treatment with CarboCell-formulated antibiotics was carried out without systemic antibiotic administration. The bacterial burden was determined by quantitative bacteriology. RESULTS: CarboCell formulations eliminated S. aureus in infected implant rat models. In the translational implant-associated pig model, surgical debridement and injection of clindamycin-releasing CarboCell formulations resulted in pathogen-free bone tissues and implants in 9 of 12 and full eradication in 5 of 12 pigs. CONCLUSIONS: Sustained release of antimicrobial agents mediated by the CarboCell technology demonstrated promising therapeutic efficacy in challenging translational models and may be beneficial in combination with the current standard of care.

Pharmacokinetics and Optimal Dosing of Levofloxacin in Children for Drug-Resistant Tuberculosis: An Individual Patient Data Meta-Analysis.

BACKGROUND: Each year 25 000-32 000 children develop rifampicin- or multidrug-resistant tuberculosis (RR/MDR-TB), and many more require preventive treatment. Levofloxacin is a key component of RR/MDR-TB treatment and prevention, but the existing pharmacokinetic data in children have not yet been comprehensively summarized. We aimed to characterize levofloxacin pharmacokinetics through an individual patient data meta-analysis of available studies and to determine optimal dosing in children. METHODS: Levofloxacin concentration and demographic data were pooled from 5 studies and analyzed using nonlinear mixed effects modeling. Simulations were performed using current World Health Organization (WHO)-recommended and model-informed optimized doses. Optimal levofloxacin doses were identified to target median adult area under the time-concentration curve (AUC)24 of 101 mg·h/L given current standard adult doses. RESULTS: Data from 242 children (2.8 years [0.2-16.8] was used). Apparent clearance was 3.16 L/h for a 13-kg child. Age affected clearance, reaching 50% maturation at birth and 90% maturation at 8 months. Nondispersible tablets had 29% lower apparent oral bioavailability compared to dispersible tablets. Median exposures at current WHO-recommended doses were below the AUC target for children weighing <24 kg and under <10 years, resulting in approximately half of the exposure in adults. Model-informed doses of 16-33 mg/kg for dispersible tablets or 16-50 mg/kg for nondispersible tablets were required to meet the AUC target without significantly exceeding the median adult Cmax. CONCLUSIONS: Revised weight-band dosing guidelines with doses of >20 mg/kg are required to ensure adequate exposure. Further studies are needed to determine safety and tolerability of these higher doses.

Treatment Outcome of Severe Respiratory Type B Tularemia Using Fluoroquinolones.

BACKGROUND: Fluoroquinolones lack approval for treatment of tularemia but have been used extensively for milder illness. Here, we evaluated fluoroquinolones for severe illness. METHODS: In an observational study, we identified case-patients with respiratory tularemia from July to November 2010 in Jämtland County, Sweden. We defined severe tularemia by hospitalization for >24 hours and severe bacteremic tularemia by Francisella tularensis subsp. holarctica growth in blood or pleural fluid. Clinical data and drug dosing were retrieved from electronic medical records. Chest images were reexamined. We used Kaplan-Meier curves to evaluate time to defervescence and hospital discharge. RESULTS: Among 67 case-patients (median age, 66 years; 81% males) 30-day mortality was 1.5% (1 of 67). Among 33 hospitalized persons (median age, 71 years; 82% males), 23 had nonbacteremic and 10 had bacteremic severe tularemia. Subpleural round consolidations, mediastinal lymphadenopathy, and unilateral pleural fluid were common on chest computed tomography. Among 29 hospitalized persons with complete outcome data, ciprofloxacin/levofloxacin (n = 12), ciprofloxacin/levofloxacin combinations with doxycycline and/or gentamicin (n = 11), or doxycycline as the single drug (n = 6) was used for treatment. One disease relapse occurred with doxycycline treatment. Treatment responses were rapid, with median fever duration 41.0 hours in nonbacteremic and 115.0 hours in bacteremic tularemia. Increased age-adjusted Charlson comorbidity index predicted severe bacteremic tularemia (odds ratio, 2.7 per score-point; 95% confidence interval, 1.35-5.41). A 78-year-old male with comorbidities and delayed ciprofloxacin/gentamicin treatment died. CONCLUSIONS: Fluoroquinolone treatment is effective for severe tularemia. Subpleural round consolidations and mediastinal lymphadenopathy were typical findings on computed tomography among case-patients in this study.

Assessing potential drug-drug interactions between clofazimine and other frequently used agents to treat drug-resistant tuberculosis.

Clofazimine is included in drug regimens to treat rifampicin/drug-resistant tuberculosis (DR-TB), but there is little information about its interaction with other drugs in DR-TB regimens. We evaluated the pharmacokinetic interaction between clofazimine and isoniazid, linezolid, levofloxacin, and cycloserine, dosed as terizidone. Newly diagnosed adults with DR-TB at Klerksdorp/Tshepong Hospital, South Africa, were started on the then-standard treatment with clofazimine temporarily excluded for the initial 2 weeks. Pharmacokinetic sampling was done immediately before and 3 weeks after starting clofazimine, and drug concentrations were determined using validated liquid chromatography-tandem mass spectrometry assays. The data were interpreted with population pharmacokinetics in NONMEM v7.5.1 to explore the impact of clofazimine co-administration and other relevant covariates on the pharmacokinetics of isoniazid, linezolid, levofloxacin, and cycloserine. Clofazimine, isoniazid, linezolid, levofloxacin, and cycloserine data were available for 16, 27, 21, 21, and 6 participants, respectively. The median age and weight for the full cohort were 39 years and 52 kg, respectively. Clofazimine exposures were in the expected range, and its addition to the regimen did not significantly affect the pharmacokinetics of the other drugs except levofloxacin, for which it caused a 15% reduction in clearance. A posteriori power size calculations predicted that our sample sizes had 97%, 90%, and 87% power at P < 0.05 to detect a 30% change in clearance of isoniazid, linezolid, and cycloserine, respectively. Although clofazimine increased the area under the curve of levofloxacin by 19%, this is unlikely to be of great clinical significance, and the lack of interaction with other drugs tested is reassuring.

Efflux pump effects on levofloxacin resistance in Mycobacterium abscessus.

Mycobacterium abscessus (M. abscessus) inherently displays resistance to most antibiotics, with the underlying drug resistance mechanisms remaining largely unexplored. Efflux pump is believed to play an important role in mediating drug resistance. The current study examined the potential of efflux pump inhibitors to reverse levofloxacin (LFX) resistance in M. abscessus. The reference strain of M. abscessus (ATCC19977) and 60 clinical isolates, including 41 M. abscessus subsp. abscessus and 19 M. abscessus subsp. massilense, were investigated. The drug sensitivity of M. abscessus against LFX alone or in conjunction with efflux pump inhibitors, including verapamil (VP), reserpine (RSP), carbonyl cyanide 3-chlorophenylhydrazone (CCCP), or dicyclohexylcarbodiimide (DCC), were determined by AlarmarBlue microplate assay. Drug-resistant regions of the gyrA and gyrB genes from the drug-resistant strains were sequenced. The transcription level of the efflux pump genes was monitored using qRT-PCR. All the tested strains were resistant to LFX. The drug-resistant regions from the gyrA and gyrB genes showed no mutation associated with LFX resistance. CCCP, DCC, VP, and RSP increased the susceptibility of 93.3% (56/60), 91.7% (55/60), 85% (51/60), and 83.3% (50/60) isolates to LFX by 2 to 32-fold, respectively. Elevated transcription of seven efflux pump genes was observed in isolates with a high reduction in LFX MIC values in the presence of efflux pump inhibitors. Efflux pump inhibitors can improve the antibacterial activity of LFX against M. abscessus in vitro. The overexpression of efflux-related genes in LFX-resistant isolates suggests that efflux pumps are associated with the development of LFX resistance in M. abscessus.

Osteomyelitis-relevant antibiotics at clinical concentrations show limited effectivity against acute and chronic intracellular S. aureus infections in osteocytes.

Osteomyelitis caused by Staphylococcus aureus can involve the persistent infection of osteocytes. We sought to determine if current clinically utilized antibiotics were capable of clearing an intracellular osteocyte S. aureus infection. Rifampicin, vancomycin, levofloxacin, ofloxacin, amoxicillin, oxacillin, doxycycline, linezolid, gentamicin, and tigecycline were assessed for their minimum inhibitory concentration (MIC) and minimum bactericidal concentrations against 12 S. aureus strains, at pH 5.0 and 7.2 to mimic lysosomal and cytoplasmic environments, respectively. Those antibiotics whose bone estimated achievable concentration was commonly above their respective MIC for the strains tested were further assayed in a human osteocyte infection model under acute and chronic conditions. Osteocyte-like cells were treated at 1×, 4×, and 10× the MIC for 1 and 7 days following infection (acute model), or at 15 and 21 days of infection (chronic model). The intracellular effectivity of each antibiotic was measured in terms of CFU reduction, small colony variant formation, and bacterial mRNA expression change. Only rifampicin, levofloxacin, and linezolid reduced intracellular CFU numbers significantly in the acute model. Consistent with the transition to a non-culturable state, few if any CFU could be recovered from the chronic model. However, no treatment in either model reduced the quantity of bacterial mRNA or prevented non-culturable bacteria from returning to a culturable state. These findings indicate that S. aureus adapts phenotypically during intracellular infection of osteocytes, adopting a reversible quiescent state that is protected against antibiotics, even at 10× their MIC. Thus, new therapeutic approaches are necessary to cure S. aureus intracellular infections in osteomyelitis.

Rapid in vitro activity of telavancin against Bacillus anthracis and in vivo protection against inhalation anthrax infection in the rabbit model.

Inhalation anthrax is the most severe form of Bacillus anthracis infection, often progressing to fatal conditions if left untreated. While recommended antibiotics can effectively treat anthrax when promptly administered, strains engineered for antibiotic resistance could render these drugs ineffective. Telavancin, a semisynthetic lipoglycopeptide antibiotic, was evaluated in this study as a novel therapeutic against anthrax disease. Specifically, the aims were to (i) assess in vitro potency of telavancin against 17 B. anthracis isolates by minimum inhibitory concentration (MIC) testing and (ii) evaluate protective efficacy in rabbits infected with a lethal dose of aerosolized anthrax spores and treated with human-equivalent intravenous telavancin doses (30 mg/kg every 12 hours) for 5 days post-antigen detection versus a humanized dose of levofloxacin and vehicle control. Blood samples were collected at various times post-infection to assess the level of bacteremia and antibody production, and tissues were collected to determine bacterial load. The animals' body temperatures were also recorded. Telavancin demonstrated potent bactericidal activity against all strains tested (MICs 0.06-0.125 µg/mL). Further, telavancin conveyed 100% survival in this model and cleared B. anthracis from the bloodstream and organ tissues more effectively than a humanized dose of levofloxacin. Collectively, the low MICs against all strains tested and rapid bactericidal in vivo activity demonstrate that telavancin has the potential to be an effective alternative for the treatment or prophylaxis of anthrax infection.

Diagnosis and Treatment of Helicobacter pylori Infection.

The last 5 years have seen major shifts in defining whom to test and how to treat Helicobacter pylori infection. Peptic ulcer has changed from a chronic disease to a one-off condition, and countries with a high incidence of gastric cancer have begun implementing population-wide screening and treatment. A proactive approach to testing and treatment of H. pylori is now recommended, including outreach to family members of individuals diagnosed with active infection as well as high-risk local populations such as immigrants from high-risk countries. Increasing antimicrobial resistance has resulted in an overall decline in treatment success, causing a rethinking of the approach to development of treatment guidelines as well as the need to adopt the principles of antibiotic usage and antimicrobial stewardship. Required changes include abandoning empiric use of clarithromycin, metronidazole, and levofloxacin triple therapies. Here, we discuss these transformations and give guidance regarding testing and use of therapies that are effective when given empirically.

P-type ATPase zinc transporter Rv3270 of Mycobacterium tuberculosis enhances multi-drug efflux activity.

Metal homeostasis is maintained by the uptake, storage and efflux of metal ions that are necessary for the survival of the bacterium. Homeostasis is mostly regulated by a group of transporters categorized as ABC transporters and P-type ATPases. On the other hand, efflux pumps often play a role in drug-metal cross-resistance. Here, with the help of antibiotic sensitivity, antibiotic/dye accumulation and semi-quantitative biofilm formation assessments we report the ability of Rv3270, a P-type ATPase known for its role in combating Mn2+ and Zn2+ metal ion toxicity in Mycobacterium tuberculosis, in influencing the extrusion of multiple structurally unrelated drugs and enhancing the biofilm formation of Escherichia coli and Mycobacterium smegmatis. Overexpression of Rv3270 increased the tolerance of host cells to norfloxacin, ofloxacin, sparfloxacin, ampicillin, oxacillin, amikacin and isoniazid. A significantly lower accumulation of norfloxacin, ethidium bromide, bocillin FL and levofloxacin in cells harbouring Rv3270 as compared to host cells indicated its role in enhancing efflux activity. Although over-expression of Rv3270 did not alter the susceptibility levels of levofloxacin, rifampicin and apramycin, the presence of a sub-inhibitory concentration of Zn2+ resulted in low-level tolerance towards these drugs. Of note, the expression of Rv3270 enhanced the biofilm-forming ability of the host cells strengthening its role in antimicrobial resistance. Therefore, the study indicated that the over-expression of Rv3270 enhances the drug efflux activity of the micro-organism where zinc might facilitate drug-metal cross-resistance for some antibiotics.

Effectiveness of Helicobacter pylori Treatments According to Antibiotic Resistance.

INTRODUCTION: Antibiotic resistance is one of the main factors that determine the efficacy of treatments to eradicate Helicobacter pylori infection. Our aim was to evaluate the effectiveness of first-line and rescue treatments against H. pylori in Europe according to antibiotics resistance. METHODS: Prospective, multicenter, international registry on the management of H. pylori (European Registry on H. pylori Management). All infected and culture-diagnosed adult patients registered in the Spanish Association of Gastroenterology-Research Electronic Data Capture from 2013 to 2021 were included. RESULTS: A total of 2,852 naive patients with culture results were analyzed. Resistance to clarithromycin, metronidazole, and quinolones was 22%, 27%, and 18%, respectively. The most effective treatment, regardless of resistance, were the 3-in-1 single capsule with bismuth, metronidazole, and tetracycline (91%) and the quadruple with bismuth, offering optimal cure rates even in the presence of bacterial resistance to clarithromycin or metronidazole. The concomitant regimen with tinidazole achieved an eradication rate of 99% (90/91) vs 84% (90/107) with metronidazole. Triple schedules, sequential, or concomitant regimen with metronidazole did not achieve optimal results. A total of 1,118 non-naive patients were analyzed. Resistance to clarithromycin, metronidazole, and quinolones was 49%, 41%, and 24%, respectively. The 3-in-1 single capsule (87%) and the triple therapy with levofloxacin (85%) were the only ones that provided encouraging results. DISCUSSION: In regions where the antibiotic resistance rate of H. pylori is high, eradication treatment with the 3-in-1 single capsule, the quadruple with bismuth, and concomitant with tinidazole are the best options in naive patients. In non-naive patients, the 3-in-1 single capsule and the triple therapy with levofloxacin provided encouraging results.

Nine months of bedaquiline, linezolid, levofloxacin, clofazimine, and cycloserine chemotherapy for rifampicin/multidrug-resistant tuberculosis: a multicenter, randomized, open-label non-inferiority trial in China.

BACKGROUND: We concurrently developed a prospective study to assess clinical outcomes among patients receiving 9-month bedaquiline (BDQ)-containing regimens, aiming to provide valuable data on the use of this short-course regimen in China. METHODS: This open-label, randomized, controlled, multicenter, non-inferiority trial was conducted at sixteen hospitals, and enrolled participants aged 18 years and older with pulmonary rifampicin/multidrug tuberculosis. Participants were randomly assigned, in a 1:1 ratio. Individuals within the standard-regimen group received 6 months of BDQ, linezolid, levofloxacin, clofazimine, and cycloserine plus 12 months of levofloxacin, and any three potentially effective drugs from clofazimine, cycloserine pyrazinamide, ethambutol and protionamide, whereas individuals within shorter-regimen group received 9 months of BDQ, linezolid, levofloxacin, clofazimine and cycloserine. The primary outcome was the percentage of participants with a composite unfavorable outcome (treatment failure, death, treatment discontinuation, or loss to follow-up) by the end of the treatment course after randomization in the modified intention-to-treat population. The noninferiority margin was 10%. This trial was registered with www.chictr.org.cn , ChiCTR2000029012. RESULTS: Between Jan 1, 2020, and Dec 31, 2023, 264 were screened and randomly assigned, 132 of 264 participants were assigned to the standard-regimen group and 132 were assigned to the shorter-regimen. Thirty-three (12.55%) of 264 participants were excluded from the modified intention-to-treat analysis. As a result, 231 participants were included in the modified intention-to-treat analysis (116 in the standard-regimen group and 115 in the shorter-regimen group).In the modified intention-to-treat population, unfavorable outcomes were reported in 19 (16.5%) of 115 participants for whom the outcome was assessable in the shorter-regimen group and 26 (22.4%) of 116 participants in the standard care group (risk difference 5.9 percentage points (97.5% CI - 5.8 to 17.5)). One death was reported in the standard-regimen group. The incidence of QTcF prolongation in the shorter-regimen group (22.6%, 26/115) was similar to the standard-regimen group (24.1%, 28/116). CONCLUSIONS: The 9-month, all-oral regimen is safe and efficacious for the treatment of pulmonary rifampicin/multidrug-resistant tuberculosis. The high incidence of QTc prolongation associated with the use of BDQ highlights the urgent need of routine electrocardiogram monitoring under treatment with BDQ-containing regimens in the Chinese population.

In vitro activity of delafloxacin against clinical levofloxacin-resistant Helicobacter pylori isolates.

BACKGROUND: Increasing antibiotic resistance in Helicobacter pylori necessitates research on new active molecules. In 2017, delafloxacin, a new fluoroquinolone with chemical properties of activity under acidic conditions, was approved for treatment of community-acquired bacterial pneumonia and acute bacterial skin and soft-tissue infections. Mutations in gyrA are responsible for fluoroquinolone resistance, but certain clinical isolates of H. pylori appear to display a dual phenotype: resistance to levofloxacin associated with very low delafloxacin MICs. OBJECTIVES: To estimate epidemiological cut-off (ECOFF) values and to identify mutations in the gyrA gene, specific to FQ resistance, without increasing the MICs of delafloxacin. METHODS: Clinical strains (n = 231) were collected in the bacteriology laboratory of Poitiers University Hospital over a 2 year period to determine the ECOFF of delafloxacin. Retrospectively, 101 clinical strains with an levofloxacin-resistant phenotype (MIC > 1 mg/L) were selected from 2018 to 2022 for delafloxacin MIC determination and QRDR (gyrA) sequencing. RESULTS: The estimated ECOFF of delafloxacin was ≤0.125 mg/L. No H. pylori isolate showed a levofloxacin-sensitive phenotype with a delafloxacin MIC of >0.125 mg/L. Among the levofloxacin-resistant H. pylori isolates, 53.5% had delafloxacin MICs of ≤0.125 mg/L. The N87I mutation was associated with dual levofloxacin/delafloxacin resistance (P < 0.001) in contrast to the N87K and D91N mutations (P > 0.05). Mutations D91G and D91Y were not associated with a delafloxacin resistance phenotype (P > 0.05). CONCLUSIONS: Delafloxacin seems to be a therapeutic alternative for levofloxacin-resistant strains with greater in vitro activity. However, further clinical/biological investigations are required to determine its efficacy in H. pylori eradication.

Comparison of the in vitro activities of delafloxacin and comparators against Staphylococcus epidermidis clinical strains involved in osteoarticular infections: a CRIOGO multicentre retrospective study.

OBJECTIVES: Staphylococcus epidermidis bone and joint infections (BJIs) on material are often difficult to treat. The activity of delafloxacin has not yet been studied on S. epidermidis in this context. The aim of this study was to assess its in vitro activity compared with other fluoroquinolones, against a large collection of S. epidermidis clinical strains. METHODS: We selected 538 S. epidermidis strains isolated between January 2015 and February 2023 from six French teaching hospitals. One hundred and fifty-two strains were ofloxacin susceptible and 386 were ofloxacin resistant. Identifications were performed by MS and MICs were determined using gradient concentration strips for ofloxacin, levofloxacin, moxifloxacin and delafloxacin. RESULTS: Ofloxacin-susceptible strains were susceptible to all fluoroquinolones. Resistant strains had higher MICs of all fluoroquinolones. Strains resistant to ofloxacin (89.1%) still showed susceptibility to delafloxacin when using the Staphylococcus aureus 2021 CA-SFM/EUCAST threshold of 0.25 mg/L. In contrast, only 3.9% of the ofloxacin-resistant strains remained susceptible to delafloxacin with the 0.016 mg/L S. aureus breakpoint according to CA-SFM/EUCAST guidelines in 2022. The MIC50 was 0.094 mg/L and the MIC90 was 0.38 mg/L. CONCLUSIONS: We showed low delafloxacin MICs for ofloxacin-susceptible S. epidermidis strains and a double population for ofloxacin-resistant strains. Despite the absence of breakpoints for S. epidermidis, delafloxacin may be an option for the treatment of complex BJI, including strains with MICs of ≤0.094 mg/L, leading to 64% susceptibility. This study underlines the importance for determining specific S. epidermidis delafloxacin breakpoints for the management of BJI on material.

Real-world efficacy of second-line therapies for Helicobacter pylori: a population-based study.

BACKGROUND: With the increasing prevalence of antibiotic resistance, real-world data on the optimal empirical second-line therapy for Helicobacter pylori are still limited. OBJECTIVES: To evaluate the real-world efficacy of various second-line therapies for H. pylori. PATIENTS AND METHODS: This was a retrospective population-based cohort study of all H. pylori-infected patients who had received the second-line treatment after the failure of primary clarithromycin triple therapy in Hong Kong between 2003 and 2018. The retreatment success rates of different second-line therapies were evaluated. RESULTS: A total of 7591 patients who received second-line treatment were included. Notably, the most commonly prescribed regimen was still clarithromycin triple therapy, but the frequency of use had decreased from 59.5% in 2003-06 to 28.7% in 2015-18. Concomitant non-bismuth quadruple therapy had emerged as the commonest regimen (from 3.3% to 43.9%). In a validation analysis, the sensitivity and specificity of retreatment-inferred second-line treatment failure were 88.3% and 97.1%, respectively. The overall success rate of second-line therapies was 73.6%. Bismuth quadruple therapy had the highest success rate of 85.6%, while clarithromycin triple therapy had the lowest success rate of 63.5%. Specifically, bismuth/metronidazole/tetracycline quadruple, metronidazole/tetracycline triple, levofloxacin/metronidazole/tetracycline quadruple, rifabutin/amoxicillin triple and amoxicillin/levofloxacin triple therapies had relatively higher success rates over 80%. Age, treatment duration, baseline conditions and first-line treatment used were associated with success rate. CONCLUSIONS: Bismuth quadruple therapy was the most effective second-line regimen for H. pylori in this real-world study. Despite a very low success rate, clarithromycin-containing triple therapies were still commonly used as second-line regimens.

Prediction of antimicrobial resistance in Klebsiella pneumoniae using genomic and metagenomic next-generation sequencing data.

OBJECTIVES: Klebsiella pneumoniae is a significant pathogen with increasing resistance and high mortality rates. Conventional antibiotic susceptibility testing methods are time-consuming. Next-generation sequencing has shown promise for predicting antimicrobial resistance (AMR). This study aims to develop prediction models using whole-genome sequencing data and assess their feasibility with metagenomic next-generation sequencing data from clinical samples. METHODS: On the basis of 4170 K. pneumoniae genomes, the main genetic characteristics associated with AMR were identified using a LASSO regression model. Consequently, the prediction model was established, validated and optimized using clinical isolate read simulation sequences. To evaluate the efficacy of the model, clinical specimens were collected. RESULTS: Four predictive models for amikacin, ciprofloxacin, levofloxacin, and piperacillin/tazobactam, initially had positive predictive values (PPVs) of 92%, 98%, 99%, 94%, respectively, when they were originally constructed. When applied to clinical specimens, their PPVs were 96%, 96%, 95%, and 100%, respectively. Meanwhile, there were negative predictive values (NPVs) of 100% for ciprofloxacin and levofloxacin, and 'not applicable' (NA) for amikacin and piperacillin/tazobactam. Our method achieved antibacterial phenotype classification accuracy rates of 95.92% for amikacin, 96.15% for ciprofloxacin, 95.31% for levofloxacin and 100% for piperacillin/tazobactam. The sequence-based prediction antibiotic susceptibility testing (AST) reported results in an average time of 19.5 h, compared with the 67.9 h needed for culture-based AST, resulting in a significant reduction of 48.4 h. CONCLUSIONS: These preliminary results demonstrated that the performance of prediction model for a clinically significant antimicrobial-species pair was comparable to that of phenotypic methods, thereby encouraging the expansion of sequence-based susceptibility prediction and its clinical validation and application.

Based on molecular docking and real-time PCR technology, the two-component system Bae SR was investigated on the mechanism of drug resistance in CRAB.

This study aimed to explore the role of the two-component system Bae SR in the mechanism of drug resistance in carbapenem-resistant A. baumannii (CRAB) using molecular docking and real-time polymerase chain reaction (PCR). The two-component system Bae SR of Acinetobacter baumannii was subjected to molecular docking with imipenem, meropenem, and levofloxacin. Antibacterial assays and fluorescence quantitative PCR were used to explore protein-ligand interactions and molecular biological resistance mechanisms related to CRAB. The analysis of the two-component system in A. baumannii revealed that imipenem exhibited the highest docking energy in Bae S at - 5.81 kcal/mol, while the docking energy for meropenem was - 4.92 kcal/mol. For Bae R, imipenem had a maximum docking energy of - 4.28 kcal/mol, compared with - 4.60 kcal/mol for meropenem. The highest binding energies for Bae S-levofloxacin and Bae R-levofloxacin were - 3.60 and - 3.65 kcal/mol, respectively. All imipenem-resistant strains had minimum inhibitory concentration (MIC) values of 16 µg/mL, whereas levofloxacin-resistant strains had MIC values of 8 µg/mL. The time-sterilization curve showed a significant decrease in bacterial colony numbers at 2 h under the action of 8 µg/mL imipenem, indicating antibacterial effects. In contrast, levofloxacin did not exhibit any antibacterial activity. Fluorescence quantitative PCR results revealed significantly increased relative expression levels of bae S and bae R genes in the CRAB group, which were 2 and 1.5 times higher than those in the CSAB group, respectively, with statistically significant differences. Molecular docking in this study found that the combination of Bae SR and carbapenem antibiotics (imipenem, meropenem) exhibited stronger affinity and stability compared with levofloxacin. Moreover, the overexpression of the two-component system genes in carbapenem-resistant A. baumannii enhanced its resistance to carbapenem, providing theoretical and practical insights into carbapenem resistance in respiratory tract infections caused by A. baumannii.

Characterization of genes related to the efflux pump and porin in multidrug-resistant Escherichia coli strains isolated from patients with COVID-19 after secondary infection.

BACKGROUND: Escherichia coli (E. coli) is a multidrug resistant opportunistic pathogen that can cause secondary bacterial infections in patients with COVID-19. This study aimed to determine the antimicrobial resistance profile of E. coli as a secondary bacterial infection in patients with COVID-19 and to assess the prevalence and characterization of genes related to efflux pumps and porin. METHODS: A total of 50 nonduplicate E. coli isolates were collected as secondary bacterial infections in COVID-19 patients. The isolates were cultured from sputum samples. Confirmation and antibiotic susceptibility testing were conducted by Vitek 2. PCR was used to assess the prevalence of the efflux pump and porin-related genes in the isolates. The phenotypic and genotypic evolution of antibiotic resistance genes related to the efflux pump was evaluated. RESULTS: The E. coli isolates demonstrated high resistance to ampicillin (100%), cefixime (62%), cefepime (62%), amoxicillin-clavulanic acid (60%), cefuroxime (60%), and ceftriaxone (58%). The susceptibility of E. coli to ertapenem was greatest (92%), followed by imipenem (88%), meropenem (86%), tigecycline (80%), and levofloxacin (76%). Regarding efflux pump gene combinations, there was a significant association between the acrA gene and increased resistance to levofloxacin, between the acrB gene and decreased resistance to meropenem and increased resistance to levofloxacin, and between the ompF and ompC genes and increased resistance to gentamicin. CONCLUSIONS: The antibiotics ertapenem, imipenem, meropenem, tigecycline, and levofloxacin were effective against E. coli in patients with COVID-19. Genes encoding efflux pumps and porins, such as acrA, acrB, and outer membrane porins, were highly distributed among all the isolates. Efflux pump inhibitors could be alternative antibiotics for restoring tetracycline activity in E. coli isolates.

Lysozyme binding with amikacin and levofloxacin studied by tritium probe, fluorescence spectroscopy and molecular docking.

Lysozyme complexes with amikacin and levofloxacin were studied by spectroscopy approaches as well as using a tritium probe. Tritium was used as a labeling agent to trace labeled compound concentration in a system of two immiscible liquids and in the atomic form to determine the possible position of the binding site. Co-adsorption of protein and drug at the liquid-liquid interface was analyzed by scintillation phase method that allowed us to directly determine the amount of protein and drug in the mixed adsorption layer. Also, tensiometric measuring of the interfacial tension was used for calculation of binding parameters accordingly to Fainerman model. The treatment of complexes with atomic tritium followed by trypsinolysis and analysis of tritium distribution in the lysozyme peptides reveals the binding sites, binding energies in which were analyzed using molecular docking. Formation of complexes with amikacin and levofloxacin preserves secondar structure of protein. However, the formation of complex with amikacin leads to the almost total loss of the enzymatic activity of lysozyme and the redshift of the maximum on the lysozyme fluorescence band. A slight decrease in the distribution coefficient of lysozyme in the presence of amikacin assumes that the complex has higher hydrophilicity in comparison to lysozyme without additives. The most favorable for binding were the positions of the active centers that included amino acids Asp52 and Glu35, as well as in the vicinity of peptide His15-Arg21, with the participation of amino acids Tyr20, Arg14. In the case of levofloxacin, the formation of lysozyme-ligand complex in aqueous solution is possible without changing the microenvironment of the active center of the protein. Binding of levofloxacin to the active center of the enzyme was the most favorable, but Asp52 and Glu35 that are responsible for the enzymatic activity of lysozyme, were not affected.

Efficacy of adding levofloxacin to gemcitabine and nanoparticle-albumin-binding paclitaxel combination therapy in patients with advanced pancreatic cancer: study protocol for a multicenter, randomized phase 2 trial (T-CORE2201).

BACKGROUND: Advanced pancreatic cancer is one of the leading causes of cancer-related deaths. For patients with advanced pancreatic cancer, gemcitabine and nanoparticle albumin-binding paclitaxel (nabPTX) combination (GEM/nabPTX) therapy is one of the recommended first-line treatments. Several retrospective studies have suggested that the addition of levofloxacin improves the efficacy of GEM/nabPTX therapy in patients with advanced pancreatic cancer. This prospective study aims to evaluate whether the addition of antibiotics improves the treatment efficacy of GEM/nabPTX as a first-line chemotherapy in patients with advanced pancreatic cancer. METHODS: This multicenter, prospective, randomized, phase 2 trial will included 140 patients. Patients with advanced pancreatic cancer will be randomized in a 1:1 ratio to either the GEM/nabPTX therapy group or the GEM/nabPTX plus levofloxacin group. The primary endpoint for the two groups is median progression-free survival time (mPFS) for the full analysis set (FAS). The secondary endpoints for the two groups are median overall survival (mOS), response rate (RR), disease control rate (DCR), and adverse event (AE) for the FAS and mPFS, mOS, RR, DCR, and AE for the per-protocol set. This study will enroll patients treated with GEM/nabPTX as the first-line chemotherapy for stage IV pancreatic adenocarcinoma. DISCUSSION: GEM/nabPTX is a standard first-line chemotherapy regimen for patients with advanced pancreatic cancer. Recently, the superiority of 5-fluorouracil, liposomal irinotecan, and oxaliplatin combination therapy (NALIRIFOX) to GEM/nabPTX as first-line therapy for pancreatic cancer has been reported. However, the efficacy of NALIRIFOX is inadequate. Based on previous retrospective studies, it is hypothesized that treatment efficacy will improve when levofloxacin is added to GEM/nabPTX therapy. If the AEs (such as leukopenia, neutropenia, and peripheral neuropathy) that occur at an increased rate with levofloxacin and GEM/nabPTX combination therapy can be carefully monitored and properly managed, this simple intervention can be expected to improve the prognosis of patients with advanced pancreatic cancer. TRIAL REGISTRATION: This study was registered with the Japan Registry of Clinical Trials (jRCT; registry number: jRCTs021230005).

Mechanochemically Induced Solid-State Transformations of Levofloxacin.

Levofloxacin hemihydrate (LVXh) is a complex fluoroquinolone drug that exists in both hydrated and anhydrous/dehydrated forms. Due to the complexity of such a compound, the primary aim of this study was to investigate the amorphization capabilities and solid-state transformations of LVXh when exposed to mechanical treatment using ball milling. Spray drying was utilized as a comparative method for investigating the capabilities of complete LVX amorphous (LVXam) formation. The solid states of the samples produced were comprehensively characterized by powder X-ray diffraction, thermal analysis, infrared spectroscopy, Rietveld method, and dynamic vapor sorption. The kinetics of the process and the quantification of phases at different time points were conducted by Rietveld refinement. The impact of the different mills, milling conditions, and parameters on the composition of the resulting powders was examined. A kinetic investigation of samples produced using both mills disclosed that it was in fact possible to partially amorphize LVXh upon mechanical treatment. It was discovered that LVXh first transformed to the anhydrous/dehydrated form γ (LVXγ), as an intermediate phase, before converting to LVXam. The mechanism of LVXam formation by ball milling was successfully revealed, and a new method of forming LVXγ and LVXam by mechanical forces was developed. Spray drying from water depicted that complete amorphization of LVXh was possible. The amorphous form of LVX had a glass transition temperature of 80 °C. The comparison of methods highlighted that the formation of LVXam is thus both mechanism- and process-dependent. Dynamic vapor sorption studies of both LVXam samples showed comparable stability properties and crystallized to the most stable hemihydrate form upon analysis. In summary, this work contributed to the detailed understanding of solid-state transformations of essential fluoroquinolones while employing greener and more sustainable manufacturing methods.