A Recombinant Thermophilic and Glucose-Tolerant GH1 ß-Glucosidase Derived from Hehua Hot Spring.

As a crucial enzyme for cellulose degradation, ß-glucosidase finds extensive applications in food, feed, and bioethanol production; however, its potential is often limited by inadequate thermal stability and glucose tolerance. In this study, a functional gene (lq-bg5) for a GH1 family ß-glucosidase was obtained from the metagenomic DNA of a hot spring sediment sample and heterologously expressed in E. coli and the recombinant enzyme was purified and characterized. The optimal temperature and pH of LQ-BG5 were 55 °C and 4.6, respectively. The relative residual activity of LQ-BG5 exceeded 90% at 55 °C for 9 h and 60 °C for 6 h and remained above 100% after incubation at pH 5.0-10.0 for 12 h. More importantly, LQ-BG5 demonstrated exceptional glucose tolerance with more than 40% activity remaining even at high glucose concentrations of 3000 mM. Thus, LQ-BG5 represents a thermophilic ß-glucosidase exhibiting excellent thermal stability and remarkable glucose tolerance, making it highly promising for lignocellulose development and utilization.

Simultaneous Determination of Levo-tetrahydropalmatine and Naltrexone in Rat Plasma by LC-MS/MS and its Application in a Pharmacokinetic Study.

BACKGROUND: Levo-tetrahydropalmatine and low-dose naltrexone are used in association with reducing cocaine-related cravings, but there are no analytical methods for the quantitative simultaneous analysis of this drug combination. OBJECTIVE: A highly selective and sensitive LC-MS/MS assay was developed and validated to simultaneously quantify l-THP and naltrexone. The analytical method for l-THP offers improved sensitivity compared to previously published methods. METHODS: The product ion transitions of l-THP and naltrexone were 357.0→193.0 and 342.2→324.1, respectively. Chromatographic separations were performed using a BEH-C18 column by an isocratic elution mode with acetonitrile and 0.1% formic acid in water containing 3 mM ammonium acetate. L-THP and naltrexone were extracted from rat plasma using a liquidliquid extraction method. RESULTS: For l-THP and naltrexone, the assay displayed good linear response over a concentration range of 0.5-1000 ng/mL and 0.25-500 ng/mL, respectively. The intra-day accuracy of the method for l-THP and naltrexone was 93.8-101% with a precision (%CV) of 2.43-8.15% and 93.4-108% with a precision of 3.47-8.22%. The inter-day accuracy for l-THP and naltrexone was 91.2-102% with a CV of 2.46-8.06% and 91.5-97.8% with a CV of 3.29-8.92%, respectively. CONCLUSION: The assay has been used for pharmacokinetic studies of l-THP and naltrexone in the rat.

Efficacy of polysaccharide iron complex in IDA rats: A comparative study with iron protein succinylate and ferrous succinate.

Iron deficiency anemia (IDA) is the most common nutrient-related health problem in the world. There is still a lack of comprehensive comparative study on the efficacies of commonly used iron supplements such as polysaccharide iron complex (PIC), iron protein succinylate (IPS) and ferrous succinate (FS) for IDA. In this study, we compared the PIC, IPS and FS efficacies in IDA rats via intragastric administration. The results showed that the three iron supplements had similar efficacies. PIC/IPS/FS at a dose of 15 mg Fe/kg/d for 10 d increased the hematological and serum biochemical parameters to 2.15/2.12/2.18 (Hb), 1.71/1.67/1.69 (RBC), 2.10/2.11/2.12 (HCT), 1.26/1.22/1.22 (MCV), all 1.34 (MCH), 1.15/1.15/1.14 (MCHC), 1.94/1.82/1.91 (SF), 9.75/9.67/9.53 (SI), and 23.30/22.68/21.64 (TS) times, and reduced TIBC to 0.42/0.43/0.44 times, compared to untreated IDA rats. PIC performed slightly better than IPS and FS in restoring MCV level. Meanwhile, the heart, spleen and kidney coefficients reduced to 67%/74%/65% (heart), all 59% (spleen) and 87%/88%/88% (kidney), and the liver coefficient increased to 116%/115%/116%, compared to untreated IDA rats. The liver iron content was found to be more affected by IDA than the spleen iron content. PIC/IPS/FS at 15 mg Fe/kg/d increased organ iron contents to 4.20/3.97/4.03 times (liver) and 1.36/1.24/1.41 times (spleen) within 10 d compared to untreated IDA rats, and PIC-H and FS were slightly better than IPS in restoring spleen iron content. The results of this study can provide useful data information for the comparison of three iron supplements, PIC, IPS and FS.

Association of serum Asprosin concentrations with heart failure.

BACKGROUND: To analyze the association of serum Asprosin concentrations with heart failure (HF). METHODS: A total of 103 patients with HF were included in the HF group, and 103 patients with health checkups were included in the non-HF group. The serum Asprosin levels of the two groups were measured, and relevant clinical data were collected for statistical analysis. RESULTS: Compared with the non-HF group, the serum Asprosin concentration was significantly higher in the HF group, and the difference was statistically significant (P < 0.001). According to the serum Asprosin levels, we divided all the subjects into three quartiles. We found that the prevalence of HF increased with increasing serum Asprosin levels in the three groups (P < 0.001). Serum Asprosin levels were positively correlated with NT-ProBNP (P < 0.05) and negatively correlated with LVEF (P < 0.001). Dichotomous logistic regression analysis found Asprosin and age to be independent risk factors for HF (OR = 1.010, 95% CI: 1.003-1.018; OR = 1.058, 95% CI:1.004-1.665, respectively). Combining Asprosin and NT-proBNP indicators to draw ROC curves can improve the specificity and sensitivity of HF diagnosis. CONCLUSIONS: Serum Asprosin levels were significantly elevated in HF patients. The serum Asprosin level is an independent risk factor for HF, and the combined detection of Asprosin and NT-proBNP levels can improve the accuracy of HF diagnosis.

Comparative metabolomics reveal key pathways associated with the synergistic activities of aztreonam and clavulanate combination against multidrug-resistant Escherichia coli.

IMPORTANCE: Multidrug-resistant Escherichia coli is a major threat to the health care system and is associated with poor outcomes in infected patients. The combined use of antibiotics has become an important treatment method for multidrug-resistant bacteria. However, the mechanism for their synergism has yet to be explored.

Cloning, Heterologous Expression, and Characterization of a Neutral Uricase from Arthrobacter sp. CSAJ-16 in Cangshan Mountain.

Uricase (or Urate oxidase), a key enzyme involved in purine metabolism, is commonly used in treating conditions such as gout, hyperuricemia, and tumor lysis syndrome. In this study, a uricase-producing strain (named CSAJ-16) was isolated from the soil sample of Cangshan Mountain, Yunnan Province, China. This strain was identified as Arthrobacter sp. CSAJ-16. Based on the gene sequence alignment, the uricase gene (named aruox) of Arthrobacter sp. CSAJ-16 was amplified and heterologously expressed. The recombinant uricase (ArUOX) was about 32 kDa. The optimal pH and temperature of ArUOX were pH 7 and 20°C, respectively. The ArUOX remained above 50% relative activity after incubation at 37°C for 100 min or at pH 6.0-8.6 for 24 h. Moreover, metal ions such as K+, Mg2+, Ca2+, Ba2+ and Pb2+ can significantly enhance the activity of ArUOX (> 200%). These enzymatic properties indicate that ArUOX has potential applications in pharmaceutical enzymes and uric acid detection kits.

Metabolomics revealed mechanism for the synergistic effect of sulbactam, polymyxin-B and amikacin combination against Acinetobacter baumannii.

Introduction: The emergence of multidrug-resistant (MDR) Acinetobacter baumannii prompts clinicians to consider treating these infections with polymyxin combination. Methods: Metabolomic analysis was applied to investigate the synergistic effects of polymyxin-B, amikacin and sulbactam combination therapy against MDR A. baumannii harboring OXA-23 and other drug resistant genes. The drug concentrations tested were based on their clinical breakpoints: polymyxin-B (2 mg/L), amikacin (16 mg/L), polymyxin-B/amikacin (2/16 mg/L), and polymyxin-B/amikacin/sulbactam (2/16/4 mg/L). Results: The triple antibiotic combination significantly disrupted levels of metabolites involved in cell outer membrane structure including fatty acids, glycerophospholipids, nucleotides, amino acids and peptides as early as 15 min after administration. Amikacin and polymyxin-B alone perturbed a large number of metabolites at 15 min and 1 h, respectively, but the changes in metabolites were short-lived lasting for less than 4 h. In contrast, the combination treatment disrupted a large amount of metabolites beyond 4 h. Compared to the double-combination, the addition of sulbactam to polymyxin-B/amikacin combination produce a greater disorder in A. baumannii metabolome that further confer susceptibility of bacteria to the antibiotics. Conclusion: The metabolomic analysis identified mechanisms responsible for the synergistic activities of polymyxin-B/amikacin/sulbactam against MDR A. baumannii.

Ultrahigh-performance liquid chromatography-high-resolution mass spectrometry-based plasma metabolomics study of thymoma and thymic hyperplasia.

RATIONALE: Thymoma is a rare malignant tumor but it is the most common primary tumor of the anterior mediastinum. The current imaging methods for thymoma screening suffer from false positive rate problems, and thymoma pathogenesis remains elusive. Study of thymoma metabolic characteristics could provide clues for improving the diagnosis and understanding the pathogenesis of thymoma. METHODS: Metabolic profiling of plasma from thymoma and thymic hyperplasia patients was performed using ultrahigh-performance liquid chromatography combined with high-resolution mass spectrometry in both positive and negative ionization modes. After pre- and post-processing, the dataset was divided into three age groups and statistical analysis was performed to select differential metabolites of thymoma. For feature identification, experimental tandem mass spectra were matched to those of databases and available chemical standards, and also manually annotated with plausible chemical structures to ensure high identification confidence. RESULTS: A total of 47 differential metabolites were identified in thymoma. Significantly higher levels of histidine, sphinganine 1-phosphate, lactic acid dimer, phenylacetylglutamine, LPC (18:3) and LPC (16:1), and significantly lower levels of phenylalanine, indole-3-propionic acid (IPA), hippuric acid and mesobilirubinogen were associated with thymoma. Tryptophan level in thymoma-associated myasthenia gravis (TAMG) was significantly lower than that of the MG(-) group. IPA and hippuric acid abundances exhibited increasing trends from indolent to aggressive thymoma. CONCLUSIONS: Our study revealed aberrant aromatic amino acid metabolism and fatty acid oxidation might be associated with thymoma. The identified unique metabolic characteristics of thymoma may provide valuable information for study of the molecular mechanism of thymoma pathogenesis, and improvement of diagnosis and discovery of new therapeutic strategies for thymoma.

The interplay between N6-methyladenosine and precancerous liver disease: molecular functions and mechanisms.

N6-methyladenosine(m6A) is one of the most abundant modifications of mammalian cellular RNAs. m6A regulates various biological functions in epitranscriptomic ways, including RNA stability, decay, splicing, translation and nuclear export. Recent studies have indicated the growing importance of m6A modification in precancerous disease, influencing viral replication, immune escape, and carcinogenesis. Here, we review the role of m6A modification in HBV/HCV infection, NAFLD and liver fibrosis, and its function in liver disease pathogenesis. Our review will provide a new sight for the innovative treatment strategy for precancerous liver disease.

Physiologically-based pharmacokinetic modeling to inform dosing regimens and routes of administration of rifampicin and colistin combination against Acinetobacter baumannii.

BACKGROUND: Carbapenem-resistant Acinetobacter baumannii (CRAB) is resistant to major antibiotics such as penicillin, cephalosporin, fluoroquinolone and aminoglycoside, and has become a significant nosocomial pathogen. The efficacy of rifampicin and colistin combination against CRAB could be dependent on the administration routes and drug concentrations at the site of infection. OBJECTIVE: The objective is to predict drug disposition in biological tissues. Treatment efficacy is extrapolated by assessing respective pharmacodynamic (PD) indices, as well as parameters associated with the emergence of resistance. METHODS: Physiologically-based pharmacokinetic models of rifampicin and colistin were utilized to predict tissue exposures. Dosing regimens and administration routes for combination therapy were evaluated in terms of in vitro antimicrobial susceptibility of A. baumannii associated with targeted PD indices and resistance parameters. RESULTS: Simulated exposures in blood, heart, lung, skin and brain were consistent with reported penetration rates. The results demonstrated that a combination of colistin and rifampicin using conventional intravenous (i.v.) doses could achieve effective exposures in the blood and skin. However, for lung infections, colistin by inhalation would be required due to low lung penetration from intravenous route. Inhaled colistin alone provided good PD coverage but this practice could encourage the emergence of additional resistance which may be overcome by a combination regimen that includes inhaled rifampicin. CONCLUSION: This in silico extrapolation provides valuable information on dosing regimens and routes of administration against CRAB infections in specific tissues. The PBPK modeling approach could be a non-invasive way to inform therapeutic benefits of combination antimicrobial therapy.

Evaluation of Vitamin D Status and the Analysis of Risk Factors of Vitamin D Deficiency in Twin Pregnancies.

OBJECTIVE: Optimization of maternal vitamin D (VD) status has beneficial effects on pregnancies, but little is known about it of twin pregnancies (TP). Our aim was to promote the current understanding of VD status and its associated factors in TP. METHODS: We performed liquid chromatography-tandem mass spectrometry to quantify 25-hydroxyvitamin D [25(OH)D] and used the enzyme-linked immunosorbent assay method to detect vitamin D binding protein (VDBP) in 218 singleton pregnancies (SP) and 236 TP. RESULTS: Levels of 25(OH)D and VDBP were higher in TP than SP. The 25(OH)D, free 25(OH)D, C-3 epimer of 25-hydroxyvitamin D [epi-25(OH)D], and VDBP all increased with gestational progress. Age, body mass index, and hemoglobin level were associated with VD deficiency (VDD). Analysis of covariance demonstrated that the 25(OH)D and VDBP of TP and SP still showed differences after adjusting for the above associated factors. CONCLUSION: Differences in VD status were found in SP and TP, suggesting that the assessment of VD status in TP should be treated with caution. High VDD prevalence is observed among pregnant Chinese women, and it is recommended to promote evaluation for VDD.

Biotransformation of Penindolone, an Influenza A Virus Inhibitor.

Penindolone (PND) is a novel broad-spectrum anti-Influenza A Virus (IAV) agent blocking hemagglutinin-mediated adsorption and membrane fusion. The goal of this work was to reveal the metabolic route of PND in rats. Ultra-high-performance liquid chromatography tandem high-resolution mass spectrometry (UHPLC-HRMS) was used for metabolite identification in rat bile, feces and urine after administration of PND. A total of 25 metabolites, including 9 phase I metabolites and 16 phase II metabolites, were characterized. The metabolic pathways were proposed, and metabolites were visualized via Global Natural Product Social Molecular Networking (GNPS). It was found that 65.24-80.44% of the PND presented in the formation of glucuronide conjugate products in bile, and more than 51% of prototype was excreted through feces. In in vitro metabolism of PND by rat, mouse and human liver microsomes (LMs) system, PND was discovered to be eliminated in LMs to different extents with significant species differences. The effects of chemical inhibitors of isozymes on the metabolism of PND in vitro indicated that CYP2E1/2C9/3A4 and UGT1A1/1A6/1A9 were the metabolic enzymes responsible for PND metabolism. PND metabolism in vivo could be blocked by UGTs inhibitor (ibrutinib) to a certain extent. These findings provided a basis for further research and development of PND.

Changes in Lipidomics, Metabolomics, and the Gut Microbiota in CDAA-Induced NAFLD Mice after Polyene Phosphatidylcholine Treatment.

Non-alcoholic fatty liver disease (NAFLD) is the most common chronic liver disease in most parts of the world. Although there is no first-line drug approved for the treatment of NAFLD, polyene phosphatidylcholine (PPC) is used by clinicians to treat NAFLD patients. This study aimed to evaluate the efficacy of PPC on a mice model of NAFLD, and to study the PPC's mechanism of action. The mice were fed a choline-deficient, L-amino acid-defined (CDAA) diet to induce NAFLD and were subsequently treated with PPC. The treatment effects were evaluated by the liver index, histopathological examination, and routine blood chemistry analyses. Lipidomics and metabolomics analyses of 54 samples were carried out using ultraperformance liquid chromatography (UPLC) coupled to a mass spectrometer to select for changes in metabolites associated with CDAA diet-induced NAFLD and the effects of PPC treatment. The intestinal flora of mice were extracted for gene sequencing to find differences before and after the induction of NAFLD and PPC treatment. PPC significantly improved the CDAA diet-induced NAFLD condition in mice. A total of 19 metabolites including 5 polar metabolites and 14 lipids showed marked changes. In addition, significant differences in the abundance of Lactobacillus were associated with NAFLD. We inferred that the protective therapeutic effect of PPC on the liver was related to the supplement of phosphatidylcholine, lysophosphatidylcholine, and sphingomyelin (PC, LPC, and SM, resectively) and acylcarnitine metabolism. This study developed a methodology for exploring the pathogenesis of NAFLD and can be extended to other therapeutic agents for treating NAFLD.

Pharmacokinetic/Pharmacodynamic Evaluation of Aztreonam/Amoxicillin/Clavulanate Combination against New Delhi Metallo-ß-Lactamase and Serine-ß-Lactamase Co-Producing Escherichia coli and Klebsiella pneumoniae.

This study aimed to examine specific niches and usage for the aztreonam/amoxicillin/clavulanate combination and to use population pharmacokinetic simulations of clinical dosing regimens to predict the impact of this combination on restricting mutant selection. The in vitro susceptibility of 19 New-Delhi metallo-ß-lactamase (NDM)-producing clinical isolates to amoxicillin/clavulanate and aztreonam alone and in co-administration was determined based on the minimum inhibitory concentration (MIC) and mutant prevention concentration (MPC). The fractions of a 24-h duration that the free drug concentration was within the mutant selection window (fTMSW) and above the MPC (fT>MPC) in both plasma and epithelial lining fluid were determined from simulations of 10,000 subject profiles based on regimens by renal function categories. This combination reduced the MIC of aztreonam and amoxicillin/clavulanate to values below their clinical breakpoint in 7/9 K. pneumoniae and 8/9 E. coli, depending on the ß-lactamase genes detected in the isolate. In the majority of the tested isolates, the combination resulted in fT>MPC > 90% and fTMSW < 10% for both aztreonam and amoxicillin/clavulanate. Clinical dosing regimens of aztreonam and amoxicillin/clavulanate were sufficient to provide mutant restriction coverage for MPC and MIC ≤ 4 mg/L. This combination has limited coverage against NDM- and extended-spectrum ß-lactamase co-producing E. coli and K. pneumoniae and is not effective against isolates carrying plasmid-mediated AmpC and KPC-2. This study offers a potential scope and limitations as to where the aztreonam/amoxicillin/clavulanate combination may succeed or fail.

Study on endogenous inhibitors against PD-L1: cAMP as a potential candidate.

The discovery of new anti-cancer drugs targeting the PD-1/PD-L1 pathway has been a research hotspot in recent years. In this study, biological affinity ultrafiltration (BAU), UPLC-HRMS, molecular dynamic (MD) simulations and molecular docking methods were applied to search for endogenous active compounds that can inhibit the binding of PD-L1 to PD-1. We screened dozens of potential cancer related endogenous compounds. Surprisingly, cyclic adenosine monophosphate (cAMP) was found to have a direct inhibitory effect on the PD-1/PD-L1 binding with an in vitro IC50 value of about 36.4 ± 9.3 µM determined by homogeneous time-resolved fluorescence (HTRF) assay. cAMP could recover the proliferation of Jurkat T cells co-cultured with DU-145 cells and may suppress PD-L1 expression of DU-145 cells. cAMP was demonstrated to bind and induce PD-L1 dimerization by FRET assay, and also predicted by MD simulations and molecular docking. The finding of cAMP as a potential inhibitor directly targeting the PD-1/PD-L1 interaction could advance our understanding of the activity of endogenous compounds regulating PD-L1.

Developing an improved UHPLC method for impurity profile analysis of ceftriaxone using analytical quality by design.

In this study, we developed a new reversed-phase ultra-high-performance liquid chromatography (UHPLC) method for comprehensively measuring impurities in ceftriaxone. The method was developed based on the Chinese Pharmacopoeia (ChP) HPLC method, which is limited by the lack of selectivity to potential impurities and a long running time. Screening experiments showed that octylamine concentration, mobile phase pH, and organic phase ratio were critical method parameters. Further optimisation and Monte-Carlo simulations were performed to map out the design space. The selected working conditions resulted in a complete separation of the impurity profile in approximately 10 min. A multivariate approach confirmed that the method was robust, and the proportion of acetonitrile should be carefully controlled. Additionally, the developed UHPLC method could be transferred back to HPLC in a single step using a Columns Calculator, providing a new approach for the rapid and effective development of the HPLC method. Our findings could serve as a reference for developing the next version of the ChP.

Risk factors for hospital-acquired pneumonia among inpatients with mental disorders in a large mental health center within a tertiary general hospital.

BACKGROUND: Few researchers have investigated the incidence of and risk factors for hospital-acquired pneumonia (HAP) among inpatients with mental disorders in a general hospital. METHODS: This study included patients with mental disorders hospitalized in a large mental health center (situated in a general hospital) between January 1, 2017, and July 31, 2021 (excluding January 1, 2020- May 31, 2020). Risk factors for HAP were identified by logistic regression analysis after propensity score matching (PSM, 1:4) for gender, age, duration of observation, and hospital ward. RESULTS: The study included 16,864 patients. HAP incidence rate was 1.15% overall, 2.11% in closed wards, 0.75% in open wards, 4.45% in patients with organic mental disorders, 1.80% in patients with schizophrenia spectrum disorders, and 0.84% in patients with mood disorders. Risk factors for HAP after PSM were hypoproteinemia, chronic liver disease, use of clozapine, hospitalization during the previous 180 days, body mass index (BMI) ≤18.5 kg/m2, cholinesterase inhibitor use, and mood stabilizer use. CONCLUSIONS: HAP was common among inpatients with mental disorders. Risk factors for HAP in patients with mental disorders include hypoproteinemia, chronic liver disease, hospitalization during the past 180 days, BMI ≤18.5 kg/m2, and use of clozapine, cholinesterase inhibitors, or mood stabilizers.

Characterization of a thermophilic and glucose-tolerant GH1 ß-glucosidase from hot springs and its prospective application in corn stover degradation.

Introduction: ß-Glucosidase serves as the pivotal rate-limiting enzyme in the cellulose degradation process, facilitating the hydrolysis of cellobiose and cellooligosaccharides into glucose. However, the widespread application of numerous ß-glucosidases is hindered by their limited thermostability and low glucose tolerance, particularly in elevated-temperature and high-glucose environments. Methods: This study presents an analysis of a ß-glucosidase gene belonging to the GH1 family, denoted lqbg8, which was isolated from the metagenomic repository of Hehua hot spring located in Tengchong, China. Subsequently, the gene was cloned and heterologously expressed in Escherichia coli BL21(DE3). Post expression, the recombinant ß-glucosidase (LQBG8) underwent purification through a Ni affinity chromatography column, thereby enabling the in-depth exploration of its enzymatic properties. Results: LQBG8 had an optimal temperature of 70°C and an optimum pH of 5.6. LQBG8 retained 100 and 70% of its maximum activity after 2-h incubation periods at 65°C and 70°C, respectively. Moreover, even following exposure to pH ranges of 3.0-10.0 for 24 h, LQBG8 retained approximately 80% of its initial activity. Notably, the enzymatic prowess of LQBG8 remained substantial at glucose concentrations of up to 3 M, with a retention of over 60% relative activity. The kinetic parameters of LQBG8 were characterized using cellobiose as substrate, with Km and Vmax values of 28 ± 1.9 mg/mL and 55 ± 3.2 µmol/min/mg, respectively. Furthermore, the introduction of LQBG8 (at a concentration of 0.03 mg/mL) into a conventional cellulase reaction system led to an impressive 43.7% augmentation in glucose yield from corn stover over a 24-h period. Molecular dynamics simulations offered valuable insights into LQBG8's thermophilic nature, attributing its robust stability to reduced fluctuations, conformational changes, and heightened structural rigidity in comparison to mesophilic ß-glucosidases. Discussion: In summation, its thermophilic, thermostable, and glucose-tolerant attributes, render LQBG8 ripe for potential applications across diverse domains encompassing food, feed, and the production of lignocellulosic ethanol.

The combination effect of meropenem/sulbactam/polymyxin-B on the pharmacodynamic parameters for mutant selection windows against carbapenem-resistant Acinetobacter baumannii.

The objective of this study was to evaluate whether combinations of sulbactam, meropenem, and polymyxin-B could reduce or close the gap of mutant selection window (MSW) of individual antibiotics against Acinetobacter baumannii harboring OXA-23. MICs of three antimicrobials used alone and in combination (meropenem/polymyxin-B or meropenem/polymyxin-B/sulbactam) were obtained in 11 clinical isolates and mutant prevention concentrations were determined in 4 of the 11 isolates. All isolates were resistant to meropenem or polymyxin-B. Combining meropenem and polymyxin-B with or without sulbactam resulted in synergistic bactericidal activities. Pharmacokinetic (PK) simulations of drug concentrations in the blood and epithelial lining fluid coupled with pharmacodynamic (PD) evaluations revealed that the fractions of time over the 24-h in terms of free drug concentration within the MSW (fTMSW) and above the MPC (fT>MPC) were optimized by combination therapy. The resultant clinical regimens of meropenem, polymyxin-B, and sulbactam evaluated in the PK-PD analysis were 2 g q8h, 2.5 mg/kg loading dose followed by 1.5 mg/kg q12h, and 3 g q8h, respectively, in patients with normal renal function. Subsequent corresponding equivalent exposure regimens would depend on the extent of renal failure. The overall results indicate that combination antibiotics consisting of sulbactam/meropenem/polymyxin-B can confer potential efficacy against A. baumannii harboring OXA-23, and reduce the opportunity for bacteria to develop further resistance. This study provides a framework for pharmacodynamic evaluation of drug-resistant mutant suppression in an antimicrobial co-administration setting. The results thereby lay the groundwork for additional studies and future clinical confirmation is warranted.

Effects of amikacin, polymyxin-B, and sulbactam combination on the pharmacodynamic indices of mutant selection against multi-drug resistant Acinetobacter baumannii.

Amikacin and polymyxins as monotherapies are ineffective against multidrug-resistant Acinetobacter baumannii at the clinical dose. When polymyxins, aminoglycosides, and sulbactam are co-administered, the combinations exhibit in vitro synergistic activities. The minimum inhibitory concentration (MIC) and mutant prevention concentration (MPC) were determined in 11 and 5 clinical resistant isolates of A. baumannii harboring OXA-23, respectively, in order to derive the fraction of time over the 24-h wherein the free drug concentration was within the mutant selection window (fTMSW) and the fraction of time that the free drug concentration was above the MPC (fT>MPC) from simulated pharmacokinetic profiles. The combination of these three antibiotics can confer susceptibility in multi-drug resistant A. baumannii and reduce the opportunity for bacteria to develop further resistance. Clinical intravenous dosing regimens of amikacin, polymyxin-B, and sulbactam were predicted to optimize fTMSW and fT>MPC from drug exposures in the blood. Mean fT>MPC were ≥ 60% and ≥ 80% for amikacin and polymyxin-B, whereas mean fTMSW was reduced to <30% and <15%, respectively, in the triple antibiotic combination. Due to the low free drug concentration of amikacin and polymyxin-B simulated in the epithelial lining fluid, the two predicted pharmacodynamic parameters in the lung after intravenous administration were not optimal even in the combination therapy setting.