Targeted inhibition of gut bacterial ß-glucuronidases by octyl gallate alleviates mycophenolate mofetil-induced gastrointestinal toxicity.

Mycophenolate mofetil (MMF) is a viable therapeutic option against various immune disorders as a chemotherapeutic agent. Nevertheless, its application has been undermined by the gastrotoxic metabolites (mycophenolic acid glucuronide, MPAG) produced by microbiome-associated ß-glucuronidase (ßGUS). Therefore, controlling microbiota-produced ßGUS underlines the potential strategy to improve MMF efficacy by overcoming the dosage limitation. In this study, the octyl gallate (OG) was identified with promising inhibitory activity on hydrolysis of PNPG in our high throughput screening based on a chemical collection of approximately 2000 natural products. Furthermore, OG was also found to inhibit a broad spectrum of BGUSs, including mini-Loop1, Loop 2, mini-Loop 2, and mini-Loop1,2. The further in vivo experiments demonstrated that administration of 20 mg/kg OG resulted in predominant reduction in the activity of BGUSs while displayed no impact on the overall fecal microbiome in mice. Furthermore, in the MMF-induced colitis model, the administration of OG at a dosage of 20 mg/kg effectively mitigated the gastrointestinal toxicity, and systematically reverted the colitis phenotypes. These findings indicate that the OG holds promising clinical potential for the prevention of MMF-induced gastrointestinal toxicity by inhibition of BGUSs and could be developed as a combinatorial therapy with MFF for better clinical outcomes.

Phentolamine Significantly Enhances Macrolide Antibiotic Antibacterial Activity against MDR Gram-Negative Bacteria.

OBJECTIVES: Multidrug-resistant (MDR) Gram-negative bacterial infections have limited treatment options due to the impermeability of the outer membrane. New therapeutic strategies or agents are urgently needed, and combination therapies using existing antibiotics are a potentially effective means to treat these infections. In this study, we examined whether phentolamine can enhance the antibacterial activity of macrolide antibiotics against Gram-negative bacteria and investigated its mechanism of action. METHODS: Synergistic effects between phentolamine and macrolide antibiotics were evaluated by checkerboard and time-kill assays and in vivo using a Galleria mellonella infection model. We utilized a combination of biochemical tests (outer membrane permeability, ATP synthesis, ΔpH gradient measurements, and EtBr accumulation assays) with scanning electron microscopy to clarify the mechanism of phentolamine enhancement of macrolide antibacterial activity against Escherichia coli. RESULTS: In vitro tests of phentolamine combined with the macrolide antibiotics erythromycin, clarithromycin, and azithromycin indicated a synergistic action against E. coli test strains. The fractional concentration inhibitory indices (FICI) of 0.375 and 0.5 indicated a synergic effect that was consistent with kinetic time-kill assays. This synergy was also seen for Salmonella typhimurium, Klebsiella pneumoniae, and Actinobacter baumannii but not Pseudomonas aeruginosa. Similarly, a phentolamine/erythromycin combination displayed significant synergistic effects in vivo in the G. mellonella model. Phentolamine added singly to bacterial cells also resulted in direct outer membrane damage and was able to dissipate and uncouple membrane proton motive force from ATP synthesis that, resulted in enhanced cytoplasmic antibiotic accumulation via reduced efflux pump activity. CONCLUSIONS: Phentolamine potentiates macrolide antibiotic activity via reducing efflux pump activity and direct damage to the outer membrane leaflet of Gram-negative bacteria both in vitro and in vivo.

Determination of 5-nitro-2-furaldehyde as marker residue for nitrofurazone treatment in farmed shrimps and with addressing the use of a novel internal standard.

We developed a significantly improved ultra-high performance liquid chromatography-tandem mass spectrometry method for determination of 5-nitro-2-furaldehyde (NF) as a surrogate using a novel internal standard for the detection of nitrofurazone. We used 2,4-dinitrophenylhydrazine derivatization and furfural as the internal standard. Derivatization was easily performed in HCl using ultrasonic manipulation for 5 min followed by liquid extraction using ethyl acetate. The samples were concentrated and purified using reverse phase and alumina cartridges in tandem. The derivatives were separated using a linear gradient elution on a C18 column with methanol and water as the mobile phase in negative ionization mode and multiple reaction monitoring. Under the optimized conditions, the calibration curves were linear from 0.2 to 20 µg/L with correlation coefficients >0.999. Mean recoveries were 80.8 to 104.4% with the intra- and inter-day relative standard deviations <15% at spiking levels of 0.1 to 10 µg/kg. The limits of detection and quantification were 0.05 and 0.1 µg/kg, respectively. This method is a robust tool for the identification and quantitative determination of NF in shrimp samples.

[Contamination Characteristics and Safety Risk Assessment of Perflurorinated Alkylated Substances in Aquatic Products from Guangzhou].

To investigate the contamination characteristics of PFASs in aquatic products, 254 samples belonging to 6 species, including fishes, crustaceans, cephalopods, etc., were collected from Guangzhou, and 23 PFASs were determined by UPLC-MS/MS. The total detection rates of the various sample species ranged from 93.5% to 100%, and the concentrations of PFASs were relatively higher in the fishes and crustaceans. 13 PFAS components were detected in the fish samples, but only 6 PFAS components were detected in haliotis samples, and obvious differences were observed among the different species sampled. PFOA and PFOS were the predominant pollutants, with detection rates of 75.6% and 76.8%. The concentrations of PFOS were in the range of 0.19-192.27 µg·kg-1, which were obviously higher than those of the other PFAS components, and their total contamination contribution factor reached the highest percentage of 35.15%. PFOS was a major contributing factor to the PFAS contamination of fishes, crustaceans, siphonopods, and processed products; however, the predominant pollutant in Haliotis samples was PFBA. The results of the risk assessment indicated that the concentrations of PFOA and PFOS would not give rise to timely harm to the consumers.

Elimination kinetics of eugenol in grass carp in a simulated transportation setting.

BACKGROUND: Fish are vulnerable to stress from over-crowding during transportation and eugenol is the most common sedative used to minimize fish injury. The ADI value of 2.5 mg/kg is recommended by the Joint FAO/WHO Expert Committee on Food Additives. The aim of this work was to study the elimination kinetics of eugenol following exposure of grass carp to a eugenol bath in a simulated transportation setting. RESULTS: Grass carp, Ctenopharyngodon idella (120 fish) were exposed for 24 h to a 10 mg/L eugenol bath. Sampling was performed during a 96 h period after the 24 h bath. Eight fish were sampled at each time point and muscle, plasma and liver concentrations of the drug were determined by ultra-performance liquid chromatography-tandem mass spectrometry. The concentration-time data of eugenol in each tissue were analyzed using non-compartmental methods. The peak concentrations (Cmax) in plasma, muscle and liver were 7.68, 5.30 and 24.63 mg/kg and the elimination half-lives (t1/2ß) were 19.79, 10.27 and 55.28 h, respectively. The clearance (CL) values were 0.10, 0.44 and 0.04 L/h/kg and the areas under the concentration-time curve (AUC0-96h) were 91.54, 22.44, and 214.12 mg·h/L in plasma, muscle and liver, respectively. After a eugenol exposure bath, drug concentrations in muscle tissue of grass carp were below 1 mg/kg at 8 h and 0.1 mg/kg at 24 h. CONCLUSIONS: The drug concentrations in muscle tissue at 8 h were lower than the recommended ADI value.

Pharmacokinetics, bioavailability and dose assessment of Cefquinome against Escherichia coli in black swans (Cygnus atratus).

BACKGROUND: The objective of this study is to investigate pharmacokinetics and dose regimens of cefquinome in black swans following intravenous (IV) and intramuscular (IM) administration at a single dose of 2 mg/kg. The MICs of cefquinome against 49 Escherichia coli isolates from black swans were determined. Monte Carlo simulation was applied to conduct the dose regimen assessment and optimization of cefquinome against E. coli in black swans, and a pharmacokinetic/pharmacodynamic (PK/PD) cutoff was established for E. coli isolates obtained in this study. RESULTS: The PK parameters of T1/2α (0.31 h), T1/2ß (1.69 h) and ClB (0.13 L/kg·h) indicated a rapid distribution and elimination of cefquinome in black swans after IV administration. After IM injection, the corresponding PK parameters of T1/2Ka, T1/2Ke, Tmax, Cmax, and F were 0.12 h, 1.62 h, 0.39 h, 5.71 µg/mL and 74.2%, respectively. The MICs of cefquinome against black swans E. coli ranged from 0.03 to 8 µg/mL, with MIC50 and MIC90 of 0.06 and 0.5 µg/mL, respectively. The PK/PD cutoff of cefquinome against E. coli was determined to be 0.2 µg/mL. Monte Carlo simulation showed that the nominal dose regimen (2 mg/kg/24 h) could not achieve a satisfactory probability of target attainment (PTA) for %TMIC ≥ 50%, indicating a risk of treatment failure and the development of potential drug resistance. CONCLUSIONS: The current daily dosage of cefquinome when divided into 12-h interval (1 mg/kg/12 h) may be effective for the treatment of E. coli infections with an MIC ≤0.5 µg/mL.

Determination of MS-222 in Water Samples by Solid-phase Extraction Coupled with Liquid Chromatography/Tandem Mass Spectrometry.

A practical solid-phase extraction (SPE) method coupled with liquid chromatography/tandem mass spectrometry (LC-MS/MS) has been developed for the determination of the fish anesthetic MS-222 in water. Water samples were concentrated and purified using three SPE cartridges of different specifications. Elution curves of MS-222 were constructed using various methanol-water solutions on the different cartridges, and SPE conditions were optimized in accordance with the elution curves. The mobile phase containing methanol and 0.1% formic acid solution with a linear gradient elution was utilized to separate MS-222 on a C18 column. Detection was carried out by a triple-quadrupole mass spectrometry with an electrospray ion source in positive mode. Recoveries of three MS-222 fortified levels of 0.05, 0.5 and 5 µg/L ranged of 82.6-101% with relative standard deviations (RSDs) below 9.36%. The limit of detection (LOD) and limit of quantification (LOQ) of MS-222 were 0.01 µg/L and 0.03 µg/L, respectively. This method was satisfactorily applied to the determination of MS-222 in actual water samples collected from aquatic product transportation vehicles or from the natural water catchments.

Low-Concentration Ciprofloxacin Selects Plasmid-Mediated Quinolone Resistance Encoding Genes and Affects Bacterial Taxa in Soil Containing Manure.

The spread of antimicrobial resistance in environment is promoted at least in part by the inappropriate use of antibiotics in animals and humans. The present study was designed to investigate the impact of different concentrations of ciprofloxacin in soil containing manure on the development of plasmid-mediated quinolone resistance (PMQR) - encoding genes and the abundance of soil bacterial communities. For these studies, high-throughput next-generation sequencing of 16S rRNA, real-time polymerase chain reaction and standard microbiologic culture methods were utilized. We demonstrated that the dissipate rate of relative abundances of some of PMQR-encoding genes, such as qnrS, oqxA and aac(6')-Ib-cr, were significantly lower with ciprofloxacin 0.04 and 0.4 mg/kg exposure as compared to no-ciprofloxacin control and ciprofloxacin 4 mg/kg exposure during 2 month. Also, the number of ciprofloxacin resistant bacteria was significantly greater in ciprofloxacin 0.04 and 0.4 mg/kg exposure as compared with no-ciprofloxacin control and the ciprofloxacin 4 mg/kg exposure. In addition, lower ciprofloxacin concentration provided a selective advantage for the populations of Xanthomonadales and Bacillales in orders while Agrobacterium, Bacillus, Enterococcus, and Burkholderia in genera. These findings suggest that lower concentration of ciprofloxacin resulted in a slower rate of PMQR-encoding genes dissipation and selected development of ciprofloxacin-resistant bacteria in soil amended with manure.

Ex vivo pharmacokinetic and pharmacodynamic analysis of valnemulin against Mycoplasma gallisepticum S6 in Mycoplasma gallisepticum and Escherichia coli co-infected chickens.

Pharmacokinetic and pharmacodynamic (PK/PD) indices against Mycoplasma gallisepticum (MG) S6 were investigated in an ex vivo PK/PD model following oral administration of valnemulin to chickens co-infected with M. gallisepticum and Escherichia coli. The minimum inhibitory concentrations (MICs) for valnemulin against MG S6 in artificial medium and chicken serum were determined. In vitro time-killing curves were established according to a series of multiples of the MIC value in an artificial medium, and ex vivo time-killing curves were established in serum samples obtained from infected chickens at different time points after oral administration with an initial titer of 1 × 10(6) color change units (CCU)/mL MG S6. The sigmoid Emax model was used to provide 24 h area under concentration-time curve/minimum inhibitory concentration ratios (AUC0-24h/MIC) for mycoplasmastasis, mycoplasmacidal activity and mycoplasmal elimination, respectively. The inoculum size and micro or macro methods exhibited little effect on MIC determination of MG, whereas matrix had a large effect. The rapid killing activity observed in in vitro time-killing curves seems to indicate that valnemulin was mycoplasmacidal and concentration dependent against MG. The AUC0-24h/MIC ratio for mycoplasmacidal activity and mycoplasmal elimination was 1321 h and 1960 h, respectively. A dosage regimen of 12.4 mg/kg/day and 18.3 mg/kg/day valnemulin was calculated for mycoplasmacidal activity and mycoplasmal elimination against MG S6, respectively.

Integration of pharmacokinetic and pharmacodynamic indices of valnemulin in broiler chickens after a single intravenous and intramuscular administration.

The antibacterial efficacy of valnemulin against Staphylococcus aureus was studied ex vivo in broiler chickens after intravenous and intramuscular administration at a dose of 10 mg/kg bodyweight (BW). The minimum inhibitory concentrations (MICs) of valnemulin against S. aureus strains ATCC 25923 in broth and serum were 0.12 and 1 µg/mL, respectively. The MIC50 and MIC90 of valnemulin against all susceptible S. aureus strains isolated from chickens in the test population were 0.06 and 0.12 µg/mL, respectively. Protein binding, which greatly influences the efficacy of valnemulin, was assayed by equilibrium dialysate in vitro. A high binding fraction of 86.2% was found, which seems in good agreement with the difference of bacterial susceptibility tests observed in broth and serum. The surrogate index of AUC0-24/MIC required for the lowest bacteriostatic effect, and 2 log10CFU reduction in bacterial count were 24.4 h and 38.0 h, respectively. The required daily dose of valnemulin for a bacteriostatic activity was calculated to be 15 mg/kg BW based on the MIC90 of 0.12 µg/mL. Considering the slow disposition process of valnemulin and an AUC0-24 h value of more than 10-fold obtained from diseased animals, a suggested dose of 3 mg/kg BW is sufficient to achieve a satisfactory therapeutic efficacy in infected broilers. Due to the time-dependent antibacterial characteristics of valnemulin, the recommended daily dose should be split into two or three sub-doses to achieve the highest effectiveness while diminishing the risk of development of bacterial resistance.

Detection of mutations in the gyrA and parC genes in Escherichia coli isolates carrying plasmid-mediated quinolone resistance genes from diseased food-producing animals.

In this study, the prevalence of plasmid-mediated quinolone resistance (PMQR) was investigated in 495 Escherichia coli isolates from diseased food-producing animals in Guangdong province, China. The quinolone resistance-determining regions (QRDRs) of the gyrA and parC genes were analysed for mutations in 55 isolates harbouring only oqxAB and all isolates harbouring other PMQR genes. Overall, 282 (57.0 %) E. coli isolates had at least one PMQR gene. oqxAB was detected in 215 isolates and predominated the PMQR genes, followed by qnrS (63 isolates), aac(6')-Ib-cr (56 isolates), qnrB (39 isolates) and qepA (18 isolates). qnrA, qnrC and qnrD were not found in any of the isolates. The rates of resistance to ciprofloxacin, enrofloxacin, levofloxacin and nalidixic acid were 75.2, 81.0, 70.5 and 97.4 %, respectively, among the 495 isolates. Eight types of mutation in gyrA were detected in 154 PMQR-positive isolates, and 147 isolates were found to have mutations in parC. PFGE analysis indicated that the PMQR-positive E. coli isolates were genetically diverse. This study demonstrated that the number of mutations in QRDRs of gyrA and/or parC was significantly associated with the MICs of quinolones (P<0.01). The rates of resistance to ciprofloxacin, enrofloxacin and nalidixic acid in PMQR-positive isolates were significantly higher than those in PMQR-negative isolates (P<0.05). In addition, the prevalence of oqxAB had significant Spearman correlation coefficients in relation to the MICs of all four tested quinolones (P<0.01).

Antimicrobial resistance, serotypes, and virulence factors of Streptococcus suis isolates from diseased pigs.

Streptococcus suis isolates from diseased pigs were examined for susceptibility to nine antimicrobials, possession of virulence-associated factors (VFs), and distribution of serotypes. The association between antimicrobial resistance (AMR) and serotypes as well as VFs was subsequently assessed. Among the isolates investigated, serotype 2 (66.04%) was mostly prevalent, followed by serotypes 1 (23.27%), 9 (1.26%), and 7 (0.63%), whereas 14 isolates were untypable by the polymerase chain reaction typing method used. Analysis with pulsed-field gel electrophoresis revealed the isolates had diverse DNA macrorestriction patterns. The frequency of antimicrobial resistance among the S. suis isolates was higher than that reported from other countries. It is notable that multiple antimicrobial resistance (three or more antimicrobials) was observed with 98.73% of the S. suis isolates, and the dominant resistance phenotype was erythromycin-tilmicosin-clindamycin-chloramphenicol-levofloxacin-ceftiofur-kanamycin-tetracycline-penicillin (35.85%). The most prevalent VFs were those encoded by muramidase-released protein (61.64%), followed by suilysin (56.60%) and extracellular factor (46.54%). Presence of VFs and the possession of certain AMR phenotypes were significantly associated as determined by statistical analysis. Together, these findings indicate that the clinical S. suis isolates obtained from diseased pigs in China are genetically diverse, are resistant to multiple antibiotics of clinical importance, and carry known virulence factors.