Detection of Multiple Antibiotic Residues in Turkish Pine and Blossom Honeys Using LC-MS/MS Method.

Due to the high demand for honey, beekeepers often feed the bees with antibiotics to protect honeybees against illnesses; the determination of veterinary drugs and their residues in bee products especially in honey is gaining importance. In this study, commercially available 15 different brands, a total of 22 honey (14 blossoms and 8 pines) samples obtained from 5 chain supermarkets in the city of Bingöl and Diyarbakir, Turkey were analysed for 29 antibiotic residues. These antibiotics belong to 10 different categories, including tetracyclines, aminoglycosides, macrolides, sulfonamides, fluoroquinolones, benzimidazoles, anthelmintic, amphenicols, quinolines, and oxazolidines. For the qualitative and quantitative determination of the antibiotics, a triple quadrupole liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used. A total of 10 out of 22 honey (8 blossom, 57.14 % and 2 pine, 25 %) samples were found to be positive for antibiotics. Among the tested antibiotics, tetracycline, dihydrostreptomycin, streptomycin, erythromycin, and sulfadimidine were detected in the honey samples. Dihydrostreptomycin and sulfadimidine were detected in 6 samples, erythromycin was determined in 4 samples, streptomycin was found in 2 samples, and lastly, tetracycline was detected only in one sample. The highest and the lowest concentrations of antibiotics detected in the samples were dihydrostreptomycin and erythromycin found at the amount of 992.58 µg/kg and 0.77 µg/kg respectively. The proposed method was validated with a limit of quantification (LOQ) and limit of detection (LOD) ranging between 0.42 and 3.22 µg /kg and 0.13-0.97 µg /kg respectively. Good linearities were also achieved ranging between R2 =0.987 and 0.999.

[Survey on the use of antibiotics among Swiss equine veterinarians]. / Umfrage zum Antibiotikagebrauch bei Schweizer Tierärzten mit Pferdeanteil.

INTRODUCTION: The aim of this study was to evaluate the current use of antibiotics by Swiss equine veterinarians and to compare the results with a similar study from 2013 before the introduction of the web tool Antibiotic Scout. The survey was sent to equine veterinarians according to the member database of the Swiss Veterinary Association (GST, SVS). The demographic data of the respondents and their antibiotics usage were collected. In addition, six different case scenarios were presented with questions to their potential antibiotic usage, active substance/preparation and the dosing scheme. The dosage provided was compared with the dosage information approved by Swissmedic in the information for healthcare professionals and the recommendations of the antibiotic scout. A backward logistic regression analysis was performed to assess the association between different aspects of antibiotic use and demographic data. The response rate was 94/739 (13 %), 22 of the 94 (23 %) had also participated in the 2013 study. 47/94 (50 %) of the respondents obtained their information from the antibiotic scout. The respondents indicated that they used an antibiotic in 16 %-88 % depending on the case scenario. Neither 3rd nor 4th generation cephalosporins or fluoroquinolones were used in the case scenarios. Dihydrostreptomycin was indicated as a possible antibiotic in a case scenario by 14/94 (15 %) of the respondents. Respondents who had already taken part in the 2013 survey used dihydrostreptomycin significantly more frequently (7/22, 32 % vs. 7/72, 10 %; p = 0,047). 29/81 (36 %) had underdosed compared to the prescribing information and 38/81 (47 %) compared to the antibiotic scout; neither was associated with demographic data. The use of non-equine-licensed antimicrobial products was directly related to the number of veterinarians in the practice (p = 0,007) and to the percentage of horses (p = 0,02). No association between demographics and peri-operative antibiotic use >24h (17/44, 39 %) was detected. The antibiotic prescribing habits of Swiss equine veterinarians have improved over the last 10 years. The antibiotic use decreased compared to the study of Schwechler et al. in 2013 by 0-16 % depending on the case scenario. The use of 3rd and 4th generation cephalosporins was reduced by 4 % and fluoroquinolones by 7 %. Underdosing according to scientific recommendations was reduced by 32 %. Furthermore, there is a need for additional information regarding the indication for antimicrobial use and the adequate use of perioperative antibiotics.

INTRODUCTION: L'objectif de cette étude était d'évaluer l'utilisation actuelle des antibiotiques par les vétérinaires équins suisses et de comparer les résultats avec une étude similaire de 2013 avant l'introduction de l'outil web AntibioticScout. L'enquête a été envoyée aux vétérinaires équins selon la base de données des membres de la Société des Vétérinaires Suisses (SVS). Les données démographiques des répondants et leur utilisation d'antibiotiques ont été collectées. En outre, six scénarios de cas différents ont été présentés avec des questions quant à l'utilisation potentielle d'antibiotiques, la substance active/préparation et le schéma posologique. La posologie fournie a été comparée à celle approuvée par Swissmedic dans les informations destinées aux professionnels de la santé et aux recommandations de l'AntibioticScout. Une analyse de régression logistique inverse a été réalisée pour évaluer l'association entre les différents aspects de l'utilisation des antibiotiques et les données démographiques. Le taux de réponse était de 94/739 (13 %), 22 des 94 (23 %) avaient également participé à l'étude de 2013. 47/94 (50 %) des répondants ont obtenu leurs informations à partir de AntibioticScout. Les répondants ont indiqué avoir utilisé un antibiotique dans 16 %-88 % selon le cas de figure. Ni les céphalosporines de 3ème ou 4ème génération ni les fluoroquinolones n'ont été utilisées dans les scénarios de cas. La dihydrostreptomycine a été indiquée comme un antibiotique possible dans un scénario de cas par 14/94 (15 %) des répondants. Les répondants qui avaient déjà participé à l>enquête de 2013 ont utilisé la dihydrostreptomycine significativement plus fréquemment (7/22, 32 % vs. 7/72, 10 % ; p = 0,047). 29/81 (36 %) avaient sous-dosé par rapport aux informations de prescription et 38/81 (47 %) par rapport à AntibioticScout ; ni l'un ni l'autre n'était associé aux données démographiques. L'utilisation de produits antimicrobiens non homologués pour les équidés était directement liée au nombre de vétérinaires dans le cabinet (p = 0,007) et au pourcentage de chevaux (p = 0,02). Aucune association entre les données démographiques et l'utilisation périopératoire d'antibiotiques >24h (17/44, 39 %) n'a été détectée. Les habitudes de prescription d'antibiotiques des vétérinaires équins suisses se sont améliorées au cours des 10 dernières années. L'utilisation d'antibiotiques a diminué par rapport à l'étude de Schwechler et al. en 2013 de 0 à 16 % selon le scénario du cas. L'utilisation des céphalosporines de 3ème et 4ème génération a été réduite de 4 % et les fluoroquinolones de 7 %. Le sous-dosage selon les recommandations scientifiques a été réduit de 32 %. Il est d'autre part nécessaire d>obtenir des informations supplémentaires concernant l>indication de l>utilisation d>antimicrobiens et l>utilisation adéquate des antibiotiques périopératoires.

Bifunctional magnetic ZnCdSe/ZnS quantum dots nanocomposite-based lateral flow immunoassay for ultrasensitive detection of streptomycin and dihydrostreptomycin in milk, muscle, liver, kidney, and honey.

In this study, bifunctional magnetic ZnCdSe/ZnS quantum dots nanocomposite (MQNs) were synthesized, and firstly used to develop a lateral flow immunoassay (LFIA) for streptomycin (STR) and dihydrostreptomycin (DHSTR) detection in milk, muscle, liver, kidney, and honey simultaneously. The fluorescence signal of MQNs was 9-fold stronger than that of the original quantum dots. The detection limits of the established MQNs-LFIA for STR and DHSTR in five samples were 0.08-1.78 µg/kg, the quantitation limits were 0.26-5.87 µg/kg, the recoveries were between 85.0% and 120.0%, and the coefficient of variations were between 0.8% and 19.3%, respectively. The sensitivity was up to 42-fold more sensitive than the reported LFIAs. The single blind test results of 25 samples were consistent with that of the confirmation method (R2 ≥ 0.99). Besides, a portable reader was self-developed and used for rapid quantification. Our study demonstrated MQNs as a promising signal-amplifying tag can be used for ultrasensitive detection of chemical contaminants in foods.

Single pot in situ aqueous derivatization and subsequent determination of streptomycin and dihydrostreptomycin residues in honey by means of mass spectrometry.

Streptomycin (STR) and dihydrostreptomycin (DSTR) are the typically encountered aminoglycoside (AMG) residues in honey. For AMG analysis, studies in literature involve impractical and expensive applications such as ion-pairing chromatography, immunoassays, pre and post column derivatizations, or SPE approaches. Pretreatments of these methods are toilsome and costly. Herein, one-pot, aqueous in-situ derivatization method was presented as a superior protocol. Time and cost-efficient UHPLC-MS/MS method has been developed, and practical sample preparation was introduced. Satisfactory results were reported in method verification studies. The mean recovery values were 102.6% for STR and 101.3% for DSTR. Average values between 1.5% and 9.9% RSDs were found at intra and inter-day precisions. CCα (5.7 and 5.8 µg/kg) and CCß (6.2 and 6.4 µg/kg) values were calculated for STR and DSTR respectively. AMG residues were found in 29 out of 110 analyzed samples using validated method. Described novelty enabled comprehensive analysis in an inexpensive and straightforward manner.

A highly sensitive and quantitative time resolved fluorescent microspheres lateral flow immunoassay for streptomycin and dihydrostreptomycin in milk, honey, muscle, liver, and kidney.

Streptomycin (STR) and dihydrostreptomycin (DHSTR) are clinically widely used in the prevention and treatment of animal diseases. Unreasonable use or abuse can easily cause adverse effects on human health. Thus, it is very necessary to establish a rapid detection method for STR and DHSTR in animal-derived foods. In this study, a time-resolved fluorescent microspheres lateral flow immunoassay (TRFM-LFIA) integrated with a portable fluorescence reader was developed for STR and DHSTR detection. The cut-off values of the TRFM-LFIA for STR and DHSTR in milk/honey/muscle/liver/kidney were both 30/15/80/25/60 µg kg-1, respectively, the limits of detection were 1.10/0.28/2.82/1.52/3.02, 0.75/0.23/1.76/1.21/2.35 µg kg-1, respectively, and the limits of quantitation were 3.62/0.93/9.31/5.02/9.96, 2.48/0.76/5.81/3.99/7.76 µg kg-1, respectively. The recoveries ranged from 80.0% to 120.1%, with coefficient of variation from 2.8% to 14.3%, respectively. The parallel experiment of 25 samples showed excellent correlation (R2 > 0.99) between ELISA kit and TRFM-LFIA. The sample pretreatment is very simple, and the results can be achieved in 8 min. This sensitive, simple, rapid and portable analysis strategy can provide universal technical support for the residue detection of veterinary drugs and even small molecular hazard factors.

Quantitative structure-activity relationships, molecular docking and molecular dynamics simulations reveal drug repurposing candidates as potent SARS-CoV-2 main protease inhibitors.

The current outbreak of COVID-19 is leading an unprecedented scientific effort focusing on targeting SARS-CoV-2 proteins critical for its viral replication. Herein, we performed high-throughput virtual screening of more than eleven thousand FDA-approved drugs using backpropagation-based artificial neural networks (q2LOO = 0.60, r2 = 0.80 and r2pred = 0.91), partial-least-square (PLS) regression (q2LOO = 0.83, r2 = 0.62 and r2pred = 0.70) and sequential minimal optimization (SMO) regression (q2LOO = 0.70, r2 = 0.80 and r2pred = 0.89). We simulated the stability of Acarbose-derived hexasaccharide, Naratriptan, Peramivir, Dihydrostreptomycin, Enviomycin, Rolitetracycline, Viomycin, Angiotensin II, Angiotensin 1-7, Angiotensinamide, Fenoterol, Zanamivir, Laninamivir and Laninamivir octanoate with 3CLpro by 100 ns and calculated binding free energy using molecular mechanics combined with Poisson-Boltzmann surface area (MM-PBSA). Our QSAR models and molecular dynamics data suggest that seven repurposed-drug candidates such as Acarbose-derived Hexasaccharide, Angiotensinamide, Dihydrostreptomycin, Enviomycin, Fenoterol, Naratriptan and Viomycin are potential SARS-CoV-2 main protease inhibitors. In addition, our QSAR models and molecular dynamics simulations revealed that His41, Asn142, Cys145, Glu166 and Gln189 are potential pharmacophoric centers for 3CLpro inhibitors. Glu166 is a potential pharmacophore for drug design and inhibitors that interact with this residue may be critical to avoid dimerization of 3CLpro. Our results will contribute to future investigations of novel chemical scaffolds and the discovery of novel hits in high-throughput screening as potential anti-SARS-CoV-2 properties.Communicated by Ramaswamy H. Sarma.

Cytotoxic effect of aminoglycoside antibiotics on the mammalian cell lines.

Aminoglycoside antibiotics have been used for treating serious but also routine infections in veterinary and human medicine for many years. The basic aim of this work is to evaluate the cytotoxicity of dihydrostreptomycin and neomycin in vitro on three cell cultures - BHK-21 (Syrian golden hamster kidney fibroblast), VERO (African green monkey kidney fibroblast) and FEA (feline embryonic fibroblast) cells. The morphological changes were examined by Giemsa staining. Cells were dried and visualized under fluorescence microscope. After the exposure to different experimental doses of dihydrostreptomycin (812.5-20000 µg/mL) and neomycin (1000-20000 µg/mL) during 24 h, the viability of BHK-21, FEA and VERO cell lines were evaluated by MTT assay. Viability of BHK-21 cells significantly (P < 0.001) decreased after treatment with 3500; 5500 and 7500 µg/mL of dihydrostreptomycin and 9000; 10000 and 20000 µg/mL of neomycin. The FEA cell viability decreased significantly (P < 0.001; P < 0.01) at 2500 and 3000 µg/mL dihydrostreptomycin and at 3000 µg/mL of neomycin treatment. Only the highest concentration of dihydrostreptomycin (20000 µg/mL) reduced VERO cell viability significantly (P < 0.01). Based on or results we can assume the effect of different antibiotics in different concentrations on cell lines is various. Detection of antibiotic toxicity to animal cells is very important because of the increasing resistance of bacteria. One of the solutions is drug dose increasing, but only to a certain concentration, since the toxic effect over the therapeutic one will prevail, which we have also shown in this work.

Role of adhesion forces in mechanosensitive channel gating in Staphylococcus aureus adhering to surfaces.

Mechanosensitive channels in bacterial membranes open or close in response to environmental changes to allow transmembrane transport, including antibiotic uptake and solute efflux. In this paper, we hypothesize that gating of mechanosensitive channels is stimulated by forces through which bacteria adhere to surfaces. Hereto, channel gating is related with adhesion forces to different surfaces of a Staphylococcus aureus strain and its isogenic ΔmscL mutant, deficient in MscL (large) channel gating. Staphylococci becoming fluorescent due to uptake of calcein, increased with adhesion force and were higher in the parent strain (66% when adhering with an adhesion force above 4.0 nN) than in the ΔmscL mutant (40% above 1.2 nN). This suggests that MscL channels open at a higher critical adhesion force than at which physically different, MscS (small) channels open and contribute to transmembrane transport. Uptake of the antibiotic dihydrostreptomycin was monitored by staphylococcal killing. The parent strain exposed to dihydrostreptomycin yielded a CFU reduction of 2.3 log-units when adhering with an adhesion force above 3.5 nN, but CFU reduction remained low (1.0 log-unit) in the mutant, independent of adhesion force. This confirms that large channels open at a higher critical adhesion-force than small channels, as also concluded from calcein transmembrane transport. Collectively, these observations support our hypothesis that adhesion forces to surfaces play an important role, next to other established driving forces, in staphylococcal channel gating. This provides an interesting extension of our understanding of transmembrane antibiotic uptake and solute efflux in infectious staphylococcal biofilms in which bacteria experience adhesion forces from a wide variety of surfaces, like those of other bacteria, tissue cells, or implanted biomaterials.

[Determination of streptomycin and dihydrostreptomycin in grapes by liquid chromatography-tandem mass spectrometry].

Streptomycin (STR) and dihydrostreptomycin (DSTR) are two of the most common aminoglycoside antibiotics used in veterinary medicine. STR is produced by some streptomyces griseus strains, and DSTR is a derivative of STR. In recent years, STR has been widely used in grapes to induce denuclearization. However, high levels of STR may have adverse effects like serious ototoxicity and nephrotoxicity. Therefore, to ensure the quality of grapes and the health of consumers, the regulation of STR and DSTR levels in grapes is required. An analytical method was developed for the identification and quantification of STR and DSTR in grapes by liquid chromatography-tandem mass spectrometry (LC-MS/MS). STR and DSTR are highly polar compounds due to the presence of various amino and hydroxyl groups in their structure. The determination of STR and DSTR poses a considerable analytical challenge, both during sample preparation and instrument analysis. In this study, the main factors governing the response, recovery, and sensitivity of these compounds, such as the type of chromatographic column, the type and proportion of the mobile phase and extraction solvent, the dosage of sodium 1-hexane sulfonate solution, and elution solvent and its volume, were investigated during sample pretreatment and instrument analysis. The STR and DSTR residues in the grape sample were extracted by ultrasonication with a phosphoric acid solution (pH 2), and cleanup and enrichment was performed using an Oasis HLB solid phase column. The analysis was performed using a UPLC Waters HSS T3 column (100 mm×2.1 mm, 1.8 µm) at the column temperature of 35℃. The injection volume was 2 µL. The mobile phase consisted of 0.1% formic acid aqueous solution and methanol with a volume ratio of 60:40. ESI-MS/MS was operated in multiple reaction monitoring (MRM) mode. External standard calibration curves were used for quantification. Based on the optimized method, both analytes displayed good linearity between 2 and 400 µg/L. The correlation coefficients were 0.9991-0.9997. Recoveries in spiked blank grape samples (5, 10, 20, and 40 µg/kg) ranged from 76.8% to 91.9%, with the relative standard deviations (RSDs) less than 10.2%, in compliance with the current legislation. The limits of detection and the limits of quantification of both analytes were 1 µg/L and 5 µg/kg, respectively. To assess the feasibility and potential of the proposed approach for routine analyses of STR and DSTR in other kinds of grape samples, the developed method was applied to the analysis of these compounds in red grapes, xinyu grapes, and xiahei grapes. The recoveries of STR and DSTR in the three kinds of blank grape samples were 77.2%-83.9% and 70.8%-78.9%, respectively, and the RSDs ranged from 3.0% to 15.6%. The results showed that the optimized methods can yield satisfactory recoveries for the analytes in grapes. In this method, the combination of Waters HSS T3 column to overcome the difficulties of the retention and separation of these highly polar compounds in the reverse phase, avoids the use of an ion-pair additive in the mobile phase to increase their retention, which is known to cause severe contamination of the column and serious ion suppression with electrospray ionization detection. In addition, the ideal enrichment and purification effect can be achieved by adding a sodium 1-hexane sulfonate solution to the superstratum extract with the use of only Oasis HLB for sample treatment. The method described herein has the advantages of easy operation, accuracy, and selectivity, making it feasible for the identification and quantification of STR and DSTR residues in grapes.

[Determination of streptomycin and dihydrostreptomycin residues in honey by hydrophilic interaction liquid chromatography-tandem mass spectrometry].

An analytical method was developed for the determination of streptomycin and dihydrostreptomycin in honey using hydrophilic interaction liquid chromatography-tandem mass spectrometry (HILIC-MS/MS). The streptomycin and dihydrostreptomycin residues in the test samples were extracted with 20 g/L trichloroacetic acid aqueous solution (including 50 mmol/L phosphate, pH 6.8) and cleaned on an Oasis HLB solid phase extraction column. The products were separated on a SIELC Obelisc R column with gradient elution using 0.5% (v/v) formic acid aqueous solution and acetonitrile as mobile phases. Streptomycin and dihydrostreptomycin were detected by liquid chromatography-tandem mass spectrometry in the positive ion mode using the external standard method. Under the optimal conditions, streptomycin and dihydrostreptomycin showed good linearity (r>0.99) in the range of 2.5-100 µg/L. The LOD and LOQ of the method was 2.0 µg/kg and 5.0 µg/kg, respectively. The spiked recoveries of the analytes from blank honey samples at the three levels of 5.0, 20.0 and 100.0 µg/kg were in the range of 86.9%-113.2% with the relative standard deviations less than 10%. With the advantages of convenience, rapidity, sensitivity and good repeatability, the method is suitable for the detection of streptomycin and dihydrostreptomycin in honey.

Investigation of the Persistence of Penicillin G and Dihydrostreptomycin Residues in Milk of Lactating Buffaloes ( Bubalus bubalis) Using Ultra-High-Performance Liquid Chromatography and Tandem Mass Spectrometry.

The purpose of this research was to evaluate the persistence of penicillin G and dihydrostreptomycin in milk of lactating buffaloes following intramuscular injection of procaine penicillin G (200000 IU/mL) and dihydrostreptomycin sulfate (250 mg/mL) every 24 h for 3 days. Milk samples were collected twice daily up to the 13th milking post-treatment and analyzed by ultra-high-performance liquid chromatography coupled to tandem mass spectrometry. The analytical method has been validated according to Commission Decision 2002/657/EC. The highest concentrations of penicillin G (275 µg kg-1) and dihydrostreptomycin (220.5 µg kg-1) were detected in the milk of the first milkings post-treatment, and levels were below the maximum residue limit of 4 and 200 µg kg-1 in all treated buffaloes at milkings 12 and 2, respectively. The results of this study demonstrate that a nine-milking withdrawal time set for bovine milk was not adequate for depletion of penicillin G in lactating buffaloes.

Dihydrostreptomycin Directly Binds to, Modulates, and Passes through the MscL Channel Pore.

The primary mechanism of action of the antibiotic dihydrostreptomycin is binding to and modifying the function of the bacterial ribosome, thus leading to decreased and aberrant translation of proteins; however, the routes by which it enters the bacterial cell are largely unknown. The mechanosensitive channel of large conductance, MscL, is found in the vast majority of bacterial species, where it serves as an emergency release valve rescuing the cell from sudden decreases in external osmolarity. While it is known that MscL expression increases the potency of dihydrostreptomycin, it has remained unclear if this effect is due to a direct interaction. Here, we use a combination of genetic screening, MD simulations, and biochemical and mutational approaches to determine if dihydrostreptomycin directly interacts with MscL. Our data strongly suggest that dihydrostreptomycin binds to a specific site on MscL and modifies its conformation, thus allowing the passage of K+ and glutamate out of, and dihydrostreptomycin into, the cell.

[Determination of streptomycin and dihydrostreptomycin residues in tomato ketchup by UPLC-MS/MS].

OBJECTIVE: To develop a method for simultaneous determination of streptomycin and dihydrostreptomycin residues in tomato ketchup by ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). METHODS: The sample was dissolved with phosphorus solution (pH 2) and extracted by ultrasonic. The pigment was removed with n-hexane. Then, the sample was cleaned up by HLB SPE. The HILIC chromatographic column (100 mm x 2.1 mm, 1.7 µm) was used to complete the separation under gradient elution. The mixed solution of 0.1% formic acid solution and acetonitrile was used as mobile phase. The detection of streptomycin and dihydrostreptomycin were carried out by MS/MS under multiple reaction monitoring (MRM) mode. The external standard method was used for quantitative analysis. RESULTS: The calibration curves for streptomycin and dihydrostreptomycin were indicated in the range of 0.005 - 0.100 mg/kg, and the detection limits were both 0.005 mg/kg. The recoveries of streptomycin and dihydrostreptomycin were ranged from 79.5% to 93.9% with relative standard deviations no more than 10%. CONCLUSION: The method is simple and accurate to meet the requirements for determination of streptomycin and dihydrostreptomycin residues in tomato ketchup.

Tmc1 Point Mutation Affects Ca2+ Sensitivity and Block by Dihydrostreptomycin of the Mechanoelectrical Transducer Current of Mouse Outer Hair Cells.

The transduction of sound into electrical signals depends on mechanically sensitive ion channels in the stereociliary bundle. The molecular composition of this mechanoelectrical transducer (MET) channel is not yet known. Transmembrane channel-like protein isoforms 1 (TMC1) and 2 (TMC2) have been proposed to form part of the MET channel, although their exact roles are still unclear. Using Beethoven (Tmc1(Bth/Bth)) mice, which have an M412K point mutation in TMC1 that adds a positive charge, we found that Ca(2+) permeability and conductance of the MET channel of outer hair cells (OHCs) were reduced. Tmc1(Bth/Bth) OHCs were also less sensitive to block by the permeant MET channel blocker dihydrostreptomycin, whether applied extracellularly or intracellularly. These findings suggest that the amino acid that is mutated in Bth is situated at or near the negatively charged binding site for dihydrostreptomycin within the permeation pore of the channel. We also found that the Ca(2+) dependence of the operating range of the MET channel was altered by the M412K mutation. Depolarization did not increase the resting open probability of the MET current of Tmc1(Bth/Bth) OHCs, whereas raising the intracellular concentration of the Ca(2+) chelator BAPTA caused smaller increases in resting open probability in Bth mutant OHCs than in wild-type control cells. We propose that these observations can be explained by the reduced Ca(2+) permeability of the mutated MET channel indirectly causing the Ca(2+) sensor for adaptation, at or near the intracellular face of the MET channel, to become more sensitive to Ca(2+) influx as a compensatory mechanism. SIGNIFICANCE STATEMENT: In the auditory system, the hair cells convert sound-induced mechanical movement of the hair bundles atop these cells into electrical signals through the opening of mechanically gated ion channels at the tips of the bundles. Although the nature of these mechanoelectrical transducer (MET) channels is still unclear, recent studies implicate transmembrane channel-like protein isoform 1 (TMC1) channels in the mammalian cochlea. Using a mutant mouse model (Beethoven) for progressive hearing loss in humans (DFNA36), which harbors a point mutation in the Tmc1 gene, we show that this mutation affects the MET channel pore, reducing its Ca(2+) permeability and its affinity for the permeant blocker dihydrostreptomycin. A number of phenomena that we ascribe to Ca(2+)-dependent adaptation appear stronger, in compensation for the reduced Ca(2+) entry.

The Role of Flies in Disseminating Plasmids with Antimicrobial-Resistance Genes Between Farms.

Dissemination of antimicrobial resistance is a major global public health concern. To clarify the role of flies in disseminating antimicrobial resistance between farms, we isolated and characterized tetracycline-resistant Escherichia coli strains isolated from flies and feces of livestock from four locations housing swine (abattoir, three farms) and three cattle farms. The percentages of isolates from flies resistant to tetracycline, dihydrostreptomycin, ampicillin, and chloramphenicol (80.8%, 61.5%, 53.8%, and 50.0%, respectively) and those from animal feces (80.5%, 78.0%, 41.5%, and 46.3%, respectively) in locations housing swine were significantly higher than those from cattle farms (p<0.05). The rates of resistance in E. coli derived from flies reflected those derived from livestock feces at the same locations, suggesting that antimicrobial resistance spreads between livestock and flies on the farms. The results of pulsed-field gel electrophoresis (PFGE) analysis showed that, with a few exceptions, all E. coli isolates differed. Two pairs of tetracycline-resistant strains harbored similar plasmids with the same tetracycline-resistance genes, although the origin (fly or feces), site of isolation, and PFGE patterns of these strains differed. Therefore, flies may disseminate the plasmids between farms. Our results suggest that flies may be involved not only in spreading clones of antimicrobial-resistant bacteria within a farm but also in the widespread dissemination of plasmids with antimicrobial resistance genes between farms.

Use of experimental design to optimize a triple-potential waveform to develop a method for the determination of streptomycin and dihydrostreptomycin in pharmaceutical veterinary dosage forms by HPLC-PAD.

An HPLC-PAD method using a gold working electrode and a triple-potential waveform was developed for the simultaneous determination of streptomycin and dihydrostreptomycin in veterinary drugs. Glucose was used as the internal standard, and the triple-potential waveform was optimized using a factorial and a central composite design. The optimum potentials were as follows: amperometric detection, E1=-0.15V; cleaning potential, E2=+0.85V; and reactivation of the electrode surface, E3=-0.65V. For the separation of the aminoglycosides and the internal standard of glucose, a CarboPac™ PA1 anion exchange column was used together with a mobile phase consisting of a 0.070 mol L(-1) sodium hydroxide solution in the isocratic elution mode with a flow rate of 0.8 mL min(-1). The method was validated and applied to the determination of streptomycin and dihydrostreptomycin in veterinary formulations (injection, suspension and ointment) without any previous sample pretreatment, except for the ointments, for which a liquid-liquid extraction was required before HPLC-PAD analysis. The method showed adequate selectivity, with an accuracy of 98-107% and a precision of less than 3.9%.

[Determination of streptomycin and dihydrostreptomycin in pollens by high performance liquid chromatography-tandem mass spectrometry].

A method was established for the determination of streptomycin (STR) and dihydrostreptomycin (DHS) in pollens based on high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). The sample was extracted and cleaned-up by a C18 solid phase extraction cartridge. The separation was carried out on a Protemix WCX-NP5 column (100 mm x 2.1 mm, 5 microm) with a gradient elution using 5% (v/v) formic acid, 20 mmol/L ammonium acetate and methanol as mobile phases. The analysis of streptomycin and dihydrostreptomycin was performed under electrospray positive ionization mode. The limits of detection (LOD, S/N = 3) and limits of quantification (LOQ, S/N = 10) for the both were 5 microg/kg and 10 microg/kg, respectively. Good linearities (r > 0.99) were achieved for the target compounds over the range of 10-200 microg/L. The recoveries at three spiked levels (10, 20, 50 microg/kg) in the blank matrices, such as pollen pini, corn pollen, camellia pollen, sunflower pollen, rape pollen and bee pollen, were from 76.8% to 100.3% with the relative standard deviations varied from 3.70% to 12.6%. The method is accurate, practical, and can be applied to most of the contaminated matrices. With this method, heptafluorobutyric acid is not required as mobile phase which is harmful to MS spectrometer.

Streptomycin potency is dependent on MscL channel expression.

The antibiotic streptomycin is widely used in the treatment of microbial infections. The primary mechanism of action is inhibition of translation by binding to the ribosome, but how it enters the bacterial cell is unclear. Early in the study of this antibiotic, a mysterious streptomycin-induced potassium efflux preceding any decrease in viability was observed; it was speculated that this changed the electrochemical gradient such that streptomycin better accessed the cytoplasm. Here we use a high-throughput screen to search for compounds targeting the mechanosensitive channel of large conductance (MscL) and find dihydrostreptomycin among the 'hits'. Furthermore, we find that MscL is not only necessary for the previously described streptomycin-induced potassium efflux, but also directly increases MscL activity in electrophysiological studies. The data suggest that gating MscL is a novel mode of action of dihydrostreptomycin, and that MscL's large pore may provide a mechanism for cell entry.

Effect of 2 herbal intramammary products on milk quantity and quality compared with conventional and no dry cow therapy.

Dry cow therapy, administered at the end of lactation, is aimed at eliminating current and preventing future intramammary (IMM) bacterial infections and typically involves intramammary administration of antibiotics. Certified organic dairies in the United States are restricted from using antibiotics and must consider an alternative therapy or no dry cow therapy. The current study compared 2 herbal products to conventional dry cow therapy and no treatment for a total of 5 treatments over 2 trials. Trial 1 was conducted over 3 yr on 1 research farm and trial 2 included 4 commercial farms plus the research herd over 2 yr. Treatments included (1) a conventional IMM antibiotic and internal teat sealant (penicillin-dihydrostreptomycin and bismuth subnitrate; CON); (2) an herbal IMM product purported to act as a teat sealant (Cinnatube, New AgriTech Enterprises, Locke, NY; CIN); (3) an herbal IMM product (Phyto-Mast, Bovinity Health LLC, Narvon, PA; P-M); (4) Phyto-Mast and Cinnatube (PC); or (5) no dry cow therapy (NT). Each treatment group was balanced by breed, lactation number, due date, herd, and year. However, the CON treatment was used only in the research herd because of the intent to avoid antibiotic usage on the other 4 farms. Comparisons among treatments included the difference between pre- and posttreatment 305-d mature equivalent milk production (trial 1), somatic cell score change from dry-off to freshening at the cow and quarter levels (trials 1 and 2), and milk microbiology change over the dry period (trial 2). We detected no significant differences among treatments for milk yield differences between the lactation following treatment and the lactation preceding treatment. Changes in somatic cell score from one lactation to the next also did not differ significantly among treatments in either trial. Cure rates were not significantly different among treatments; only 19.6% of all quarters were infected at dry off. The proportion of quarters with new infections at 3 to 5d postcalving did not significantly differ among treatments, except between CIN and NT. Percentages (least squares means ± standard error) of quarters with new infections were 24 ± 21% for CON, 15 ± 7% for CIN, 30 ± 10% for P-M, 32 ± 11% for PC, and 35 ± 11% for NT. The efficacy of the herbal products was similar to that of conventional therapy, and the herbal products had no apparent adverse effects.

Transduction without tip links in cochlear hair cells is mediated by ion channels with permeation properties distinct from those of the mechano-electrical transducer channel.

Tip links between adjacent stereocilia are believed to gate mechano-electrical transducer (MET) channels and mediate the electrical responses of sensory hair cells. We found that mouse auditory hair cells that lack tip links due to genetic mutations or exposure to the Ca(2+) chelator BAPTA can, however, still respond to mechanical stimuli. These MET currents have unusual properties and are predominantly of the opposite polarity relative to those measured when tip links are present. There are other striking differences, for example, the channels are usually all closed when the hair cell is not stimulated and the currents in response to strong stimuli can be substantially larger than normal. These anomalous MET currents can also be elicited early in development, before the onset of mechano-electrical transduction with normal response polarity. Current-voltage curves of the anomalous MET currents are linear and do not show the rectification characteristic of normal MET currents. The permeant MET channel blocker dihydrostreptomycin is two orders of magnitude less effective in blocking the anomalous MET currents. The findings suggest the presence of a large population of MET channels with pore properties that are distinct from those of normal MET channels. These channels are not gated by hair-bundle links and can be activated under a variety of conditions in which normal tip-link-mediated transduction is not operational.