Bimetal MOFs catalyzed Fenton-like reaction for dual-mode detection of thiamphenicol.

In this work, we used a simple ultrasonic stripping method to synthesize a bimetal MOFs at room temperature as a nanoenzyme with peroxidase-like (POD-like) activity. Through bimetal MOFs catalytic Fenton-like competitive reaction, thiamphenicol can be quantitatively dual-mode detected by fluorescence and colorimetry. It realized the sensitive detection of thiamphenicol in water, and the limits of detection (LOD) were 0.030 nM and 0.031 nM, and the liner ranges were 0.1-150 nM and 0.1-100 nM, respectively. The methods were applied to river water, lake water and tap water samples, and with satisfactory recoveries between 97.67% and 105.54%.

Oxalated zero valent iron enables highly efficient heterogeneous Fenton reaction by self-adapting pH and accelerating proton cycle.

Heterogeneous Fenton reactions of zero-valent iron (ZVI) requires the sufficient release of Fe(II) to catalyze the H2O2 decomposition. However, the rate-limiting step of proton transfer through the passivation layer of ZVI restricted the Fe(II) release via Fe0 core corrosion. Herein we modified the shell of ZVI with highly proton-conductive FeC2O4·2H2O by ball-milling (OA-ZVIbm), and demonstrated its high heterogeneous Fenton performance of thiamphenicol (TAP) removal, with 500 times enhancement of the rate constant. More importantly, the OA-ZVIbm/H2O2 showed little attenuation of the Fenton activity during 13 successive cycles, and was applicable across a wide pH range of 3.5-9.5. Interestingly, the OA-ZVIbm/H2O2 reaction showed pH self-adapting ability, which initially reduced and then sustained the solution pH in the range of 3.5-5.2. The abundant intrinsic surface Fe(II) of OA-ZVIbm (45.54% vs. 27.52% in ZVIbm, according to Fe 2p XPS profiles) was oxidized by H2O2 and hydrolyzed to generate protons, and the FeC2O4·2H2O shell favored the fast transfer of protons to inner Fe0, therefore, the consumption-regeneration cycle of protons were accelerated to drove the production of Fe(II) for Fenton reactions, demonstrated by the more prominent H2 evolution and nearly 100% H2O2 decomposition by OA-ZVIbm. Furthermore, the FeC2O4·2H2O shell was stable and slightly decreased from 1.9% to 1.7% after the Fenton reaction. This study clarified the significance of proton transfer on the reactivity of ZVI, and provided an efficient strategy to achieve the highly efficient and robust heterogeneous Fenton reaction of ZVI for pollution control.

Molecular Mechanism of Chloramphenicol and Thiamphenicol Resistance Mediated by a Novel Oxidase, CmO, in Sphingomonadaceae.

Antibiotic resistance mediated by bacterial enzyme inactivation plays a crucial role in the degradation of antibiotics in the environment. Chloramphenicol (CAP) resistance by enzymatic inactivation comprises nitro reduction, amide bond hydrolysis, and acetylation modification. However, the molecular mechanism of enzymatic oxidation of CAP remains unknown. Here, a novel oxidase gene, cmO, was identified and confirmed biochemically. The encoded CmO oxidase could catalyze the oxidation at the C-1' and C-3' positions of CAP and thiamphenicol (TAP) in Sphingobium sp. strain CAP-1. CmO is highly conserved in members of the family Sphingomonadaceae and shares the highest amino acid similarity of 41.05% with the biochemically identified glucose methanol choline (GMC) oxidoreductases. Molecular docking and site-directed mutagenesis analyses demonstrated that CAP was anchored inside the protein pocket of CmO with the hydrogen bonding of key residues glycine (G) 99, asparagine (N) 518, methionine (M) 474, and tyrosine (Y) 380. CAP sensitivity tests demonstrated that the acetyltransferase and CmO could enable a higher level of resistance to CAP than the amide bond-hydrolyzing esterase and nitroreductase. This study provides a better theoretical basis and a novel diagnostic gene for understanding and assessing the fate and resistance risk of CAP and TAP in the environment. IMPORTANCE Rising levels of antibiotic resistance are undermining ecological and human health as a result of the indiscriminate usage of antibiotics. Various resistance mechanisms have been characterized-for example, genes encoding proteins that degrade antibiotics-and yet, this requires further exploration. In this study, we report a novel gene encoding an oxidase involved in the inactivation of typical amphenicol antibiotics (chloramphenicol and thiamphenicol), and the molecular mechanism is elucidated. The findings provide novel data with which to understand the capabilities of bacteria to tackle antibiotic stress, as well as the complex function of enzymes in the contexts of antibiotic resistance development and antibiotic removal. The reported gene can be further employed as an indicator to monitor amphenicol's fate in the environment, thus benefiting risk assessment in this era of antibiotic resistance.

Effects of natural nanoparticles on the acute toxicity, chronic effect, and oxidative stress response of phenicol antibiotics in Daphnia magna.

Natural nanoparticles (NNP) are ubiquitous in natural water and can interact with other contaminants, causing ecotoxic effects on aquatic nontarget organisms. However, the impact of NNPs on the ecotoxicity of antibiotics remains largely unknown. This work investigated the acute toxicity, chronic effect, and oxidative response and damage in Daphnia magna co-exposed to phenicol antibiotics (chloramphenicol, thiamphenicol) and different concentrations of NNPs (10 mg/L: environmentally relevant concentration; 100 mg/L: a high concentration that caused no apparent immobilization in D. magna). The results showed that the acute toxicity of chloramphenicol was increased by 10 mg/L NNPs but decreased by 100 mg/L NNPs; both concentrations of NNPs increased and decreased acute toxicities of thiamphenicol and chloramphenicol + thiamphenicol treatments, respectively. After long-term exposure, phenicol antibiotics (1 µg/L) and NNP (10 mg/L) mixtures in environmentally relevant concentrations significantly affected the reproduction of D. magna but did not influence their growth. The catalase activity, reduced glutathione level, and malonaldehyde content in D. magna also varied with the NNPs concentrations. Notably, the lowest concentration of thiamphenicol and chloramphenicol + thiamphenicol combined with NNPs significantly increased the malondialdehyde content in D. magna compared with the control, indicating membrane lipid peroxidation occurred in daphnids. This study suggests that the toxic effects of contaminants and NNPs on aquatic organisms should be considered thoroughly to avoid underestimating the hazard of these pollutants in the actual aquatic environment.

Facile Synthesis of N, S-Doped Carbon Quantum Dots from Food Waste as Fluorescent Probe for Sensitive Detection of Thiamphenicol and Its Analogues in Real Food Samples along with an Application in Bioimaging.

Herein, N, S co-doped carbon quantum dots (N, S-CDs) with high absolute quantitative yield (Abs-QY) of 50.2% were produced by hydrothermal treatment of food residue crayfish shells. A new detection method of thiamphenicol (TAP) and its analogues was established by discovering the obvious fluorescence response between TAP and N, S-CDs, which achieved a wide linear range of 20-300 µg·L-1 with a detection limit (LOD) of 11.12 µg·L-1. This novel probe exhibited strong sensitivity and shows rapid response in complex food matrices (overall detection time is less than 45 min) mainly induced by static quenching. Spiked food sample recovery ranged from 97.3 to 99.34%. Further, the cell experiments of N, S-CDs were conducted, and the cell viability remained 91.76% under high concentration of N, S-CDs due to the environmentally friendly materials. The low cytotoxicity and good cytocompatibility make these N, S-CDs compatible for cell bioimaging and intracellular detection of TAP.

Down/up-conversion dual-mode ratiometric fluorescence imprinted sensor embedded with metal-organic frameworks for dual-channel multi-emission multiplexed visual detection of thiamphenicol.

The establishment of a fluorescence sensing system for sensitive and selective visual detection of trace antibiotics is of great significance to food safety and human health risk assessment. A simple and rapid one-pot strategy was developed successfully to synthesize a down/up-conversion dual-excitation multi-emission fluorescence imprinted sensor for dual-channel thiamphenicol (TAP) detection. In this strategy, the metal-organic frameworks were in situ incorporated into the fluorescence imprinted sensor, guiding the coordination induced emission of abiotic carbon dots and signal-amplification effect of fluorescence sensing. Under dual-excitation (370 nm and 780 nm), the fluorescence imprinted sensor exhibited a dual-channel fluorescence response toward TAP with two-part linear ranges of 5.0 nM-6.0 µM and 6.0 µM-26.0 µM. Significantly, the fluorescence color ranged from blue to purple to red can be observed with the naked eye. The results of the dual-channel TAP determination in actual samples by the fluorescence imprinted sensor indicated that the fluorescence imprinted sensor provided a sensitive, selective, and multiplexed visual detection of TAP in complex sample.

Past and Present of Electrochemical Sensors and Methods for Amphenicol Antibiotic Analysis.

Amphenicols are broad-spectrum antibiotics. Despite their benefits, they also present toxic effects and therefore their presence in animal-derived food was regulated. Various analytical methods have been reported for their trace analysis in food and environmental samples, as well as in the quality control of pharmaceuticals. Among these methods, the electrochemical ones are simpler, more rapid and cost-effective. The working electrode is the core of any electroanalytical method because the selectivity and sensitivity of the determination depend on its surface activity. Therefore, this review offers a comprehensive overview of the electrochemical sensors and methods along with their performance characteristics for chloramphenicol, thiamphenicol and florfenicol detection, with a focus on those reported in the last five years. Electrode modification procedures and analytical applications of the recently described devices for amphenicol electroanalysis in various matrices (pharmaceuticals, environmental, foods), together with the sample preparation methods were discussed. Therefore, the information and the concepts contained in this review can be a starting point for future new findings in the field of amphenicol electrochemical detection.

Urinary concentrations of amphenicol antibiotics in relation to biomarkers of oxidative DNA and RNA damage in school children.

Previous studies implied that elevated exposure to amphenicol antibiotics may induce increased oxidative stress. However, the effects of amphenicol antibiotics exposure on oxidative stress damage in human have not been well studied. This study examined the associations between amphenicol antibiotics exposure and oxidative damage biomarkers in school children. Three major amphenicols including chloramphenicol (CAP), thiamphenicol (TAP), florfenicol (FF) and two biomarkers of 8-hydroxydeoxyguanosine (8-OHdG) for oxidative DNA damage and 8-oxo-7,8- dihydroguanosine (8-OHG) for oxidative RNA damage were measured in 414 morning urine samples collected from 70 school children in Shanghai, China. School children were exposed to CAP, TAP, and FF with median concentrations of 1.37, 0.36, and 0.06 µg/g Cre, respectively. Linear mixed models revealed that an interquartile range (IQR) increase of urinary TAP was positively associated with 7.75%(95% CI: 4.40%, 11.1%) increase of 8-OHdG and 7.48%(95% CI: 2.49%, 15.6%) increase of 8-OHG, respectively; in addition, CAP was associated with elevated 8-OHdG. Although FF was not found to be significantly associated with either 8-OHdG or 8-OHG, it is warranted to further investigate FF and its metabolites levels in relation to oxidative stress in future study. Our findings provide new evidence for the effects of exposure to TAP and CAP on nucleic acid oxidative damage in Children.

Hydrophilic molecularly imprinted membranes based on GO-loading for simultaneously selective recognition and detection of three amphenicols drugs in pork and milk.

It plays an important role to effective detection of amphenicols antibiotic residues in food and an important issue considering of possible impact on human health. In this work, the molecularly imprinted membranes (MIMs) were proposed to simultaneously recognize and detect thiamphenicol (TAP), florfenicol (FF) and chloramphenicol (CAP) in pork and milk samples. The synergistic effect of graphene oxide (GO), double functional monomer (methacrylate and acrylamide) and "click chemistry" strategy prompted the membranes to possess good surface hydrophilicity (48.6°), excellent selectivity and capacity to exclude macromolecules. The theoretical models of selectivity mechanism showed the selective recognition depended mainly on the hydrogen bond interaction and van der Waals interaction between the analytes and monomers. The limit of detection for 3 analytes were 0.04-0.28 µg kg-1, and showed a good correlation (r > 0.9949). Finally, this study established an effective MIMs-UHPLC-MS/MS method with great potential for the monitor of antibiotics residue in complicated matrices.

Effect of Carbonyl Cyanide Chlorophenylhydrazone on Intrabacterial Concentration and Antimicrobial Activity of Amphenicols against Swine Resistant Actinobacillus pleuropneumoniae and Pasteurella multocida.

Effects and mechanism of carbonyl cyanide chlorophenylhydrazone (CCCP) on antimicrobial activity of florfenicol (FF) and thiamphenicol (TAP) were investigated against amphenicol-resistant Actinobacillus pleuropneumoniae and Pasteurella multocida isolated from diseased swine. Broth microdilution and time-kill assays indicated that CCCP dose-dependently and substantially (4-32 fold MIC reduction) improved amphenicol antimicrobial activity. When combined with CCCP at the lowest literature reported dose (2-5 µg/mL), 85% FF resistant A. pleuropneumoniae and 92% resistant P. multocida showed significantly reduced FF MICs (≥ 4-fold). In contrast, none or few of the susceptible A. pleuropneumoniae and P. multocida had FF MICs reduction ≥ 4-fold. 90% FF resistant A. pleuropneumoniae and 96% resistant P. multocida carried the floR gene, indicating strong association with the FloR efflux pump. With CCCP, the intracellular FF concentration increased by 71% in floR+ resistant A. pleuropneumoniae and 156% in floR+ resistant P. multocida strains but not the susceptible strains. The degree of reduction in TAP MICs was found consistently in parallel to FF for both bacteria. Taken together, partially attributed to blockage of drug-efflux, the combination of FF or TAP with CCCP at sub-cytotoxic concentrations was demonstrated and showed feasibility to combat amphenicol-resistant A. pleuropneumoniae and P. multocida isolated from diseased swine.

New insights into thiamphenicol biodegradation mechanism by Sphingomonas sp. CL5.1 deciphered through metabolic and proteomic analysis.

Biological treatment is an efficient and economical process to remove thiamphenicol (TAP) residues from the environment. The discovery of TAP-degrading bacteria and the decryption of its biodegradation mechanism will be beneficial to enhance the biological removal of TAP. In this study, Sphingomonas sp. CL5.1 was found to be capable of catabolizing TAP as the sole carbon, nitrogen, and energy source. This strain could degrade 93.9% of 25 mg/L TAP in 36 h, and remove about 11.9% of the total organic carbon of TAP. A novel metabolism pathway of TAP was constructed, and the enzymes involved in TAP metabolism in strain CL5.1 were predicted via proteomic and metabolic analysis. TAP was proposed to be transformed to O-TAP via oxidation of C3-OH and DD-TAP via dehydration of C3-OH and dehydrogenation of C1-OH. A novel glucose-methanol-choline (GMC) family oxidoreductase CapO was predicted to be involved in the oxidation of C3-OH. O-TAP was supposed to be further cleaved into DCA, glycine, and PMB. Glycine might be a pivotal direct nitrogen source for strain CL5.1, and it could be involved in nitrogen metabolism through the glycine cleavage system or directly participate in the biosynthetic processes.

Maximum levels of cross-contamination for 24 antimicrobial active substances in non-target feed. Part 7: Amphenicols: florfenicol and thiamphenicol.

The specific concentrations of florfenicol and thiamphenicol in non-target feed for food-producing animals, below which there would not be an effect on the emergence of, and/or selection for, resistance in bacteria relevant for human and animal health, as well as the specific antimicrobial concentrations in feed which have an effect in terms of growth promotion/increased yield, were assessed by EFSA in collaboration with EMA. Details of the methodology used for this assessment, associated data gaps and uncertainties, are presented in a separate document. To address antimicrobial resistance, the Feed Antimicrobial Resistance Selection Concentration (FARSC) model developed specifically for the assessment was applied. The FARSC for florfenicol was estimated. However, due to the lack of data, the calculation of the FARSC for thiamphenicol was not possible until further experimental data become available. To address growth promotion, data from scientific publications obtained from an extensive literature review were used. Levels in feed that showed to have an effect on growth promotion/increased yield were reported for florfenicol, whilst for thiamphenicol no suitable data for the assessment were available. Uncertainties and data gaps associated to the levels reported were addressed. For florfenicol, it was recommended to perform further studies to supply more diverse and complete data related to the requirements for calculation of the FARSC, whereas for thiamphenicol, the recommendation was to generate the data required to fill the gaps which prevented the FARSC calculation.

Single and ternary competitive adsorption-desorption and degradation of amphenicol antibiotics in three agricultural soils.

The widespread usage of veterinary antibiotics results in antibiotic contamination and increases environmental risks. This study was evaluated the single and ternary competitive adsorption-desorption and degradation of three amphenicol antibiotics (AMs): chloramphenicol (CAP), thiamphenicol (TAP), and florfenicol (FF) in three agricultural soils. The adsorption capacity of amphenicol antibiotics in the soil was weak, and the Kf value was in the range of 0.15-3.59 µg1-1/nL1/n kg-1. In the single adsorption-desorption experiment, the ranked order of adsorption capacity was TAP > FF > CAP. However, in the ternary competitive adsorption experiment, the order was changed to be CAP > FF > TAP. The degradation of AMs in soils was performed at various conditions. All AMs were vulnerable to microbial degradation in soils. A higher initial concentration would reduce the degradation rate and enhance the persistence of AMs in soil. The degradation of AMs was positively influenced by changes in soil moisture content and culture temperatures up to 30 °C and decreased at higher temperatures. An equation was used to predict the leachability of AMs in soils and assess their risk to the water environment. The weak adsorption capacity and poor persistence of FF indicated that it may have a strong effect on groundwater based on the equation. It is imperative to further assess the biological impacts of FF at environmentally relevant concentrations given its mobility and extensive use in the livestock industry.

Evaluation of the Acute Effects and Oxidative Stress Responses of Phenicol Antibiotics and Suspended Particles in Daphnia magna.

Suspended particles (SP) exist widely in various water systems and are able to adsorb other pollutants in water, producing ecotoxic effects on aquatic nontarget species. Until now, however, few studies have focused on the effects of SP on antibiotics. Therefore, the present study investigated the effects of the mixtures of SP and phenicol antibiotics (chloramphenicol [CAP], thiamphenicol [TAP]) on acute toxicity and oxidative stress responses in Daphnia magna. The results indicated that the acute toxicity of phenicol antibiotics in D. magna was increased when combined with SP. Besides, the immobilization of daphnids caused by phenicol drugs in the presence of 10 mg/L of SP was more intense than that with 200 mg/L of SP. Furthermore, the impact of SP with diverse concentrations on the activity of catalase and the level of reduced glutathione in D. magna was different. Notably, almost all CAP + TAP + SP treatments markedly increased malondialdehyde content in D. magna, causing potential cellular oxidative damage in D. magna. In summary, the present study provides insights into the toxic effects of phenicol antibiotic and SP mixtures on aquatic organisms. Environ Toxicol Chem 2021;40:2463-2473. © 2021 SETAC.

Efficient removal of antibiotic thiamphenicol by pulsed discharge plasma coupled with complex catalysis using graphene-WO3-Fe3O4 nanocomposites.

Pulsed discharge plasma (PDP) induced complex catalysis for synergetic removal of thiamphenicol (TAP) was investigated using graphene-WO3-Fe3O4 nanocomposites. The prepared samples were characterized systematically in view of the structure and morphology, chemical bonding state, optical property, electrochemical property and magnetic property. Based on characterization and TAP degradation, the catalytic performance followed: graphene-WO3-Fe3O4>graphene-WO3>WO3, and the highest removal efficiency and kinetic constant could reached 99.3% and 0.070 min-1, respectively. With increase of catalyst dosage, the removal efficiency firstly enhanced and then declined. Lower pH value was beneficial for TAP degradation. The prepared graphene-WO3-Fe3O4 owed higher stability and lower dissolution rate of iron ion. The rGO-WO3-Fe3O4 could decompose O3 and H2O2 into more ·OH in PDP system. The degradation intermediates were characterized by fluorescence spectrograph, LC-MS and IC. Based on the detected intermediates and discrete Fourier transform (DFT) analysis, degradation pathway of TAP was proposed. Besides, the toxicity of intermediates was predicted. Finally, catalytic degradation mechanism of TAP by PDP with graphene-WO3-Fe3O4 was summarized.

Simultaneous Quantification of Chloramphenicol, Thiamphenicol, Florfenicol, and Florfenicol Amine in Animal and Aquaculture Products Using Liquid Chromatography-Tandem Mass Spectrometry.

The accumulation of antimicrobial residues in edible animal products and aquaculture products could pose health concerns to unsuspecting consumers. Hence, this study aimed to develop a validated method for simultaneous quantification of chloramphenicol (CAP), thiamphenicol (TAP), florfenicol (FF), and florfenicol amine (FFA) in beef, pork, chicken, shrimp, eel, and flatfish using a quick, easy, cheap, effective, rugged, and safe (QuEChERS) extraction method coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS). Primary-secondary amine (PSA) and MgSO4 were used for sample purification. The analytes were separated on a reversed-phase analytical column. The coefficients of determination for the linear matrix-matched calibration curves were ≥0.9941. Recovery rates ranged between 64.26 and 116.51% for the four analytes with relative standard deviations (RSDs) ≤ 18.05%. The calculated limits of detection (LODs) and limits of quantification (LOQs) were 0.005-3.1 and 0.02-10.4 µg/kg, respectively. The developed method was successfully applied for monitoring samples obtained from local markets in Seoul, Republic of Korea. The target residues were not detected in any tested matrix. The designed method was versatile, sensitive, and proved suitable for quantifying residues in animal-derived products.

[Systemic and local antibiotic therapy for acute sinusitis]. / Sistemnaya i mestnaya antibakterial'naya terapiya ostrogo sinusita.

The purpose of the study was to summarize data on modern antibiotic therapy for acute sinusitis, the role and place of topical antibacterial drugs, in particular Fluimucil-Antibiotic, in modern treatment strategies for this disease. METHODS: Search in the PUBMED electronic database (articles and related abstracts) for the keywords «acute sinusitis", «antibiotics¼, «thiamphenicol glycinate acetylcysteine¼ «biofilm¼, «respiratory tract infection¼, «N-acetylcysteine¼. RESULTS: The published research results indicate the high antibacterial activity of the Fluimucil-Antibiotic, in particular, for the topical drug use in the form of inhalations, applications, irrigation, and instillations. The published research results indicate a wide spectrum of antimicrobial action of Fluimucil-Antibiotic, its ability to destroy biofilms and prevent their formation, good pharmacokinetics, safety, which makes it possible to consider it as a potential treatment option for acute sinusitis in everyday practice.

Pharmacokinetics of thiamphenicol in Japanese quails (Coturnix japonica) after single intravenous and oral administrations.

Thiamphenicol (TP) pharmacokinetics were studied in Japanese quails (Coturnix japonica) following a single intravenous (IV) and oral (PO) administration at 30 mg/kg BW. Concentrations of TP were determined with HPLC and were analyzed by a noncompartmental method. After IV injection, elimination half-life (t1/2λz ), total body clearance (Cltot ) volume of distribution at steady state (Vdss ), and mean residence time (MRT) of TP were 3.83 hr, 0.19 L/hr/kg, 0.84 L/kg, and 4.37 hr, respectively. After oral administration of TP, the peak plasma concentration (Cmax ) was 19.81 µg/ml and was obtained at 2.00 hr (tmax ) postadministration. Elimination half-life (t1/2λz ) and mean absorption time (MAT) were 4.01 hr and 1.56 hr, respectively. The systemic bioavailability following oral administration of TP was 78.10%. TP therapy with an oral dosage of 30 mg/kg BW is suggested for a beneficial clinical effect in quails.

Improved antibacterial efficiency of inhaled thiamphenicol dry powders: Mathematical modelling of in vitro dissolution kinetic and in vitro antibacterial efficacy.

Thiamphenicol (TAP) is reported to be effective against many respiratory pathogens including methicillin-resistant Staphylococcus aureus (MRSA). However, its poor solubility in water remains as one of the obstacles hindering the preparation of inhalable TAP formulations. The aim of this study was to improve the dissolution rate of TAP by micronization, and investigate whether variations in the dissolution rates of TAP would affect its in vitro antibacterial activity. Inhalable dry powders composed of TAP microcrystals (MDP) or nanocrystals (NDP) were prepared by using a wet ball milling method followed by spray drying. The morphology, solid state and in vitro dissolution of these dry powders were characterized. In vitro antibacterial activities of the inhalable TAP dry powders against a MRSA strain were evaluated. A dissolution-efficacy model relating antibacterial activity with time and dissolution rate was established via modified time-kill assays. Upon being spray dried, the volumetric mean diameters of MDP and NDP were found to be around 5 µm. Solid state analyses showed that MDP and NDP possess the same crystalline form as the raw materials. NDP exhibited faster in vitro dissolution rate as compared to MDP. The in vitro antibacterial efficiency of NDP and MDP were superior to raw TAP when the test was performed at a TAP concentration of 32 mg/L. Simulated colony forming units predictions were consistent with the result measured in the time-kill experiments with Raw TAP, MDP and NDP. This study characterized the effect of the dissolution rate of TAP dry powders on in vitro antibacterial activity against MRSA, and an enhanced antibacterial activity of TAP was observed with an increase in the dissolution rate of TAP from the dry powders at certain concentration ranges.

Development of both type I-B and type II CRISPR/Cas genome editing systems in the cellulolytic bacterium Clostridium thermocellum.

The robust lignocellulose-solubilizing activity of C. thermocellum makes it a top candidate for consolidated bioprocessing for biofuel production. Genetic techniques for C. thermocellum have lagged behind model organisms thus limiting attempts to improve biofuel production. To improve our ability to engineer C. thermocellum, we characterized a native Type I-B and heterologous Type II Clustered Regularly-Interspaced Short Palindromic Repeat (CRISPR)/cas (CRISPR associated) systems. We repurposed the native Type I-B system for genome editing. We tested three thermophilic Cas9 variants (Type II) and found that GeoCas9, isolated from Geobacillus stearothermophilus, is active in C. thermocellum. We employed CRISPR-mediated homology directed repair to introduce a nonsense mutation into pyrF. For both editing systems, homologous recombination between the repair template and the genome appeared to be the limiting step. To overcome this limitation, we tested three novel thermophilic recombinases and demonstrated that exo/beta homologs, isolated from Acidithiobacillus caldus, are functional in C. thermocellum. For the Type I-B system an engineered strain, termed LL1586, yielded 40% genome editing efficiency at the pyrF locus and when recombineering machinery was expressed this increased to 71%. For the Type II GeoCas9 system, 12.5% genome editing efficiency was observed and when recombineering machinery was expressed, this increased to 94%. By combining the thermophilic CRISPR system (either Type I-B or Type II) with the recombinases, we developed a new tool that allows for efficient CRISPR editing. We are now poised to enable CRISPR technologies to better engineer C. thermocellum for both increased lignocellulose degradation and biofuel production.