Core-shell structure microcapsules with dual pH-responsive drug release function.

We report dual pH-responsive microcapsules manufactured by combining electrostatic droplets (ESD) and microfluidic droplets (MFD) techniques to produce monodisperse core (alginate)-shell (chitosan) structure with dual pH-responsive drug release function. The fabricated core-shell microcapsules were size controllable by tuning the synthesis parameters of the ESD and MFD systems, and were responsive in both acidic and alkaline environment, We used two model drugs (ampicillin loaded in the chitosan shell and diclofenac loaded in the alginate core) for drug delivery study. The results show that core-shell structure microcapsules have better drug release efficiency than respective core or shell particles. A biocompatibility test showed that the core-shell structure microcapsules presented positive cell viability (above 80%) when evaluated by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The results indicate that the synthesized core-shell microcapsules were a potential candidate of dual-drug carriers.

Response of bacterial isolates from Antarctic shallow sediments towards heavy metals, antibiotics and polychlorinated biphenyls.

The response of bacterial isolates from Antarctic sediments to polychlorinated biphenyls (Aroclor 1242 mixture), heavy metal salts (cadmium, copper, mercury and zinc) and antibiotics (ampicillin, chloramphenicol, kanamycin and streptomycin) was investigated. Overall, the ability to growth in the presence of Aroclor 1242 as a sole carbon source was observed for 22 isolates that mainly belonged to Psychrobacter spp. Tolerance to the heavy metals assayed in this study was in the order of Cd > Cu > Zn > Hg and appeared to be strictly related to the metal concentrations, as determined during previous chemical surveys in the same area. With regards to antibiotic assays, the response of the isolates to the tested antibiotics ranged from complete resistance to total susceptibility. In particular, resistances to ampicillin and chloramphenicol were very pronounced in the majority of isolates. Our isolates differently responded to the presence of toxic compounds primarily based on their phylogenetic affiliation and secondarily at strain level. Moreover, the high incidence of resistance either to metal or antibiotics, in addition to the capability to grow on PCBs, confirm that bacteria are able to cope and/or adapt to the occurrence pollutants even in low human-impacted environments.

The influence of pH and dissolved organic carbon on the ecotoxicity of ampicillin and clarithromycin.

The impacts of water chemistry properties including pH and dissolved organic carbon (DOC) on the ecotoxicity of active pharmaceutical ingredients (APIs) are increasingly evident. These impacts are a result of alterations in API bioavailability: pH regulates the bioavailability of many ionizable APIs via chemical speciation, whereas DOC interacts with several APIs to inhibit the APIs from traversing the membrane system of organisms. In this study, we examined the influences of pH and DOC on the bioavailability of ampicillin (AMP) and clarithromycin (CLA) with the help of a bioavailability model. The effects on bioavailability were quantified by ecotoxicity observed in cyanobacteria growth inhibition tests with Microcystis aeruginosa PCC7806. The median effect concentration (96 h-EC50total) of AMP increased by 5-fold when pH raised from 7.4 to 9.0, suggesting the zwitterionic AMP+/- species being higher in bioavailability than the negatively charged AMP- species. CLA ecotoxicity showed no significant pH-dependency, suggesting CLA+ and CLA0 species to be equally bioavailable, albeit it correlated significantly with M. aeruginosa growth rate in negative controls. In addition, DOC demonstrated no significant effects on the ecotoxicity of AMP or CLA. Overall, together with earlier results on ciprofloxacin, our data show that bioavailability relations with pH and DOC are variable among different antibiotics. Factors other than chemical speciation alone could play a role in their bioavailability, such as their molecular size and polarity.

Analyses of gene expression and physiological changes in Microcystis aeruginosa reveal the phytotoxicities of three environmental pollutants.

When the concentrations of ampicillin (Amp), atrazine (Atr) and cadmium chloride (Cd) reach excessive quantities, they become toxic to aquatic organisms. Due to the acceleration of the industrialization and the intensification of human activities, the incidence and concentrations of these types of pollutants in aquatic systems are increasing. The primary purpose of this study was to evaluate the short-term effects of Amp, Atr and Cd on the physiological indices and gene expression levels in Microcystis aeruginosa. These three pollutants significantly induced antioxidant activity but continuously accelerated the cellular oxidative damage in microalgae, which suggests an imbalance between the oxidant and the antioxidant systems. Amp, Atr and Cd also decreased the transcription of psaB, psbD1 and rbcL; the lowest transcription of these genes was only 38.1, 23.7 and 7% of the control, respectively. These three pollutants affected nitrogen (N) and phosphorous (P) uptake by inhibiting the transcription of N or P absorbing and transporting related genes, and they down regulated the transcription of microcystin-related genes, which caused a decrease of microcystin levels; and the lowest level of microcystin was only 42.4% of the control. Our results suggest that these pollutants may cause pleiotropic effects on algal growth and physiological and biochemical reactions, and they may even affect secondary metabolic processes.

Horizontal gene transfer of stress resistance genes through plasmid transport.

The horizontal gene transfer of plasmid-determined stress tolerance was achieved under lab conditions. Bacterial isolates, Enterobacter cloacae (DGE50) and Escherichia coli (DGE57) were used throughout the study. Samples were collected from contaminated marine water and soil to isolate bacterial strains having tolerance against heavy metals and antimicrobial agents. We have demonstrated plasmid transfer, from Amp(+)Cu(+)Zn(-) strain (DGE50) to Amp(-)Cu(-)Zn(+) strain (DGE57), producing Amp(+)Cu(+)Zn(+) transconjugants (DGE(TC50→57)) and Amp(+)Cu(-)Zn(+) transformants (DGE(TF50→57)). DGE57 did not carry any plasmid, therefore, it can be speculated that zinc tolerance gene in DGE57 is located on chromosome. DGE50 was found to carry three plasmids, out of which two were transferred through conjugation into DGE57, and only one was transferred through transformation. Plasmid transferred through transformation was one out of the two transferred through conjugation. Through the results of transformation it was revealed that the genes of copper and ampicillin tolerance in DGE50 were located on separate plasmids, since only ampicillin tolerance genes were transferred through transformation as a result of one plasmid transfer. By showing transfer of plasmids under lab conditions and monitoring retention of respective phenotype via conjugation and transformation, it is very well demonstrated how multiple stress tolerant strains are generated in nature.

Thermally activated persulfate oxidation of ampicillin: Kinetics, transformation products and ecotoxicity.

The heat-activated persulfate system showed encouraging results for the destruction of the widely used antibiotic Ampicillin (AMP). AMP removal follows exponential decay, and the observed kinetic constant was enhanced with persulfate (PS) dosage at the range 50-500 mg L-1 and temperature (40-60 °C), while AMP thermolysis at 60 °C was almost negligible. The apparent activation energy was estimated to 124.7 kJ mol-1. Alkaline conditions, water matrix constituents like bicarbonates, humic acid, and real water matrices retarded AMP oxidation. Experiments performed with tert-butanol and methanol as scavengers demonstrated the contribution of sulfate radicals as the dominant reactive species. Seven transformation products (TPs) of AMP have been identified from AMP destruction. An EC50 value equal to 187 mg L-1 was calculated for 72 h of exposure of the microalgae Chlorella sorokiniana to AMP. According to the ecotoxicity experiments that conducted after treatment of AMP with PS for different reaction times, no important inhibition of microalgae was noticed for contact time of 72 h and 10 d. These results indicate the formation of no toxic AMP by-products for the applied experimental conditions.

From damaged genome to cell surface: transcriptome changes during bacterial cell death triggered by loss of a restriction-modification gene complex.

Genetically programmed cell deaths play important roles in unicellular prokaryotes. In postsegregational killing, loss of a gene complex from a cell leads to its descendants' deaths. With type II restriction-modification gene complexes, such death is triggered by restriction endonuclease's attacks on under-methylated chromosomes. Here, we examined how the Escherichia coli transcriptome changes after loss of PaeR7I gene complex. At earlier time points, activation of SOS genes and sigma(E)-regulon was noticeable. With time, more SOS genes, stress-response genes (including sigma(S)-regulon, osmotic-, oxidative- and periplasmic-stress genes), biofilm-related genes, and many hitherto uncharacterized genes were induced, and genes for energy metabolism, motility and outer membrane biogenesis were repressed. As expected from the activation of sigma(E)-regulon, the death was accompanied by cell lysis and release of cellular proteins. Expression of several sigma(E)-regulon genes indeed led to cell lysis. We hypothesize that some signal was transduced, among multiple genes involved, from the damaged genome to the cell surface and led to its disintegration. These results are discussed in comparison with other forms of programmed deaths in bacteria and eukaryotes.

Probiotics protect against gut dysbiosis associated decline in learning and memory.

Gut-Brain-Axis imbalance due to gut dysbiosis by antibiotics may lead to neurobehavioral changes. Here we determine neuroprotective effect of probiotic against gut dysbiosis associated decline in learning and memory. Oral Ampicillin was used to induce gut dysbiosis while probiotic was administered along with antibiotic as treatment in Swiss albino mice. Antibiotic decreased Lactobacillus, Bifidobacterium, Firmicutes and Clostridium level. This was followed by reduced cognition, hippocampal neuronal density, intestinal crypt depth, villus length and increased corticohippocampal acetylcholinesterase, myeloperoxidase activity and oxidative stress which were partially reversed by probiotic treatment. Probiotic protected hippocampal neurons from gut dysbiosis associated inflammatory and oxidative damage in mice.

Sonocatalytic removal of ampicillin by Zn(OH)F: Effect of operating parameters, toxicological evaluation and by-products identification.

Zinc hydroxyfluoride (Zn(OH)F) sonocatalyst was prepared by using solvothermal method and was characterized by using various techniques. The sonocatalytic degradation of ampicillin (AMP) in water by sonolysis, bare ZnO and Zn(OH)F was investigated in terms of AMP removal, mineralization, detoxification of solution, and remaining by-products at the end of process. Results revealed that the sonocatalytic performance of Zn(OH)F was significantly greater than that of bare ZnO. Under the optimum conditions, the removal percentage of AMP by Zn(OH)F was ∼97% after 90 min reaction, while 51% and 36% COD and TOC removal were achieved after 120 min reaction, respectively. The study of Zn(OH)F stability revealed that the degradation efficiency of AMP was reduced by only 5% even after being reused for four experiments. The toxicity of initial and treated solutions was assessed by using agar-well diffusion method and ToxTrak™ toxicity assay, and the results indicated a substantial reduction in the toxicity of solution after the treatment. The formation of some by-products during the sonolysis and sonocatalysis was evaluated by LC-HR-MS/MS method. LC-HR-MS/MS results showed that the concentration of most by-products, which were produced after 90 min treatment by US/Zn(OH)F process, was considerably lower than those obtained during sonolysis and US/ZnO processes.

Combined effects of binary antibiotic mixture on growth, microcystin production, and extracellular release of Microcystis aeruginosa: application of response surface methodology.

The interactive effects of binary antibiotic mixtures of spiramycin (SP) and ampicillin (AMP) on Microcystis aeruginosa (MA) in terms of growth as well as microcystin production and extracellular release were investigated through the response surface methodology (RSM). SP with higher 50 and 5% effective concentrations in MA growth was more toxic to MA than AMP. RSM model for toxic unit approach suggested that the combined toxicity of SP and AMP varied from synergism to antagonism with SP/AMP mixture ratio decreasing from reversed equitoxic ratio (5:1) to equitoxic ratio (1:5). Deviations from the prediction of concentration addition (CA) and independent action (IA) model further indicated that combined toxicity of target antibiotics mixed in equivalent ratio (1:1) varied from synergism to antagonism with increasing total dose of SP and AMP. With the increase of SP/AMP mixture ratio, combined effect of mixed antibiotics on MA growth changed from stimulation to inhibition due to the variation of the combined toxicity and the increasing proportion of higher toxic component (SP) in the mixture. The mixture of target antibiotics at their environmentally relevant concentrations with increased total dose and SP/AMP mixture ratio stimulated intracellular microcystin synthesis and facilitated MA cell lysis, thus leading to the increase of microcystin productivity and extracellular release.

Antibiotic amoxicillin induces DNA lesions in mammalian cells possibly via the reactive oxygen species.

Amoxicillin is a commonly prescribed drug for anti- bacterial infection. In this study, we are interested in the effect of the drug on the cellular DNA integrity. Amoxicillin was added to the human or hamster cells in culture, and the DNA lesions induced by the drug were assessed by a comet assay with nuclear extract incubation (Wang et al., 2005 Anal Biochem 337: 70-75). Amoxicillin at 5mM rapidly induced DNA lesions in human AGS cells. The level of DNA lesions attained a maximum at about 1h, and then declined steadily and reached almost the basal level at 6h following the drug treatment. Similar induction pattern of DNA lesions was found with amoxicillin-related antibiotics such as ampicillin but not with the unrelated antibiotics such as kanamycin. For studying the repair kinetics, the cells were treated with amoxicillin for only 1h and continued culture in the absence of the drug for a certain period of time before subsequent analysis. Repair of the amoxicillin-induced DNA lesions was essentially completed within 4h. Such repair may not involve nucleotide excision repair (NER) pathway because the repair was completed with similar kinetics in both NER proficient Chinese hamster CHO-K1 cells and its isogenic NER deficient UV24 cells. Instead, the repair may involve base excision repair (BER) pathway because immunodepletion of OGG1/2, glycosylases involved in BER rendered the nuclear extract unable to excise DNA lesions induced by amoxicillin in the modified comet assay. Furthermore, amoxicillin induced intracellular reactive oxygen species (ROS) at the tempo similar to that of DNA lesions induction. Thus, we hypothesize that amoxicillin causes oxidative DNA damage in mammalian cells via ROS.

Use of a standardized JaCVAM in vivo rat comet assay protocol to assess the genotoxicity of three coded test compounds; ampicillin trihydrate, 1,2-dimethylhydrazine dihydrochloride, and N-nitrosodimethylamine.

As part of the Japanese Center for the Validation of Alternative Methods (JaCVAM)-initiative international validation study of the in vivo rat alkaline comet assay (comet assay), our laboratory examined ampicillin trihydrate (AMP), 1,2-dimethylhydrazine dihydrochloride (DMH), and N-nitrosodimethylamine (NDA) using a standard comet assay validation protocol (v14.2) developed by the JaCVAM validation management team (VMT). Coded samples were received by our laboratory along with basic MSDS information. Solubility analysis and range-finding experiments of the coded test compounds were conducted for dose selection. Animal dosing schedules, the comet assay processing and analysis, and statistical analysis were conducted in accordance with the standard protocol. Based upon our blinded evaluation, AMP was not found to exhibit evidence of genotoxicity in either the rat liver or stomach. However, both NDA and DMH were observed to cause a significant increase in % tail DNA in the rat liver at all dose levels tested. While acute hepatoxicity was observed for these compounds in the high dose group, in the investigators opinion there were a sufficient number of consistently damaged/measurable cells at the medium and low dose groups to judge these compounds as genotoxic. There was no evidence of genotoxicity from either NDA or DMH in the rat stomach. In conclusion, our laboratory observed increased DNA damage from two blinded test compounds in rat liver (later identified as genotoxic carcinogens), while no evidence of genotoxicity was observed for the third blinded test compound (later identified as a non-genotoxic, non-carcinogen). This data supports the use of a standardized protocol of the in vivo comet assay as a cost-effective alternative genotoxicity assay for regulatory testing purposes.

Cephalosporin and carbacephem nephrotoxicity. Roles of tubular cell uptake and acylating potential.

Three beta-lactams, desacetylcephaloglycin, ampicillin, and loracarbef, were studied to test a hypothesis derived from retrospective analysis of previously studied cephalosporins: that beta-lactam nephrotoxicity develops in approximate proportion to tubular cell antibiotic concentrations and lactam ring reactivities. Concentrations of each beta-lactam (and insulin) in rabbit renal cortex and serum were measured at the end of 0.5-hr infusions of 100 mg antibiotic/kg body weight and 0.5 to 0.67 hr later. Total cortical AUCs (total areas under the curve of concentration and time in renal cortex) and transported cortical AUCs (total minus insulin-space beta lactam) were calculated from these measurements. Reactivities, determined by the rate constants of lactam-ring opening at pH 10, were taken from the literature. Nephrotoxicity was quantified by grades of proximal tubular cell necrosis and by serum creatinine concentrations 2 days after infusion of 100-1500 mg/kg of the antibiotics. Desacetylcephaloglycin was slightly less nephrotoxic than cephaloglycin; the AUCs reactivities, and toxicities of these two cephalosporins fit the proposed model, particularly when allowance is made for hepatic and renal deacetylation of cephaloglycin. The very low AUCs, limited reactivity, and absence of nephrotoxicity of ampicillin also fit the model. Loracarbef had a transported AUC less than three times, and reactivity one-thirtieth, those of cefaclor, respectively. Although only at 1500 mg/kg, loracarbef was significantly more nephrotic than cefaclor. If the relativity of loracarbef with its targeted bacterial proteins, which is essentially the same as that of cefaclor, is considered instead of the base hydrolysis rate constant, than loracarbef also fits the model. By the same analysis, the comparatively high in vitro stability of other carbacephems, although pharmaceutically convenient, may not limit their nephrotoxicity.

Bacterial translocation, intestinal microflora and morphological changes of intestinal mucosa in experimental models of Clostridium difficile infection.

Bacteraemia and subsequent sepsis is one possible complication of Clostridium difficile infection. The aim of this study was to examine a correlation between bacterial translocation with morphological changes of intestinal mucosa and shifts of intestinal microflora in experimental models of C. difficile infection. A mouse model was used to study post-antibiotic shifts and mild C. difficile infection, and hamsters were used to study fatal enterocolitis. The influence of pro- and pre-biotics (lactobacilli and xylitol) were also studied in the hamster model. The quantitative composition of luminal and mucosal microflora was evaluated in different intestinal loci, inflammatory changes of mucosa were estimated in histological sections and bacterial translocation was detected in samples from blood, liver, spleen and mesenteric lymph nodes. In cases of mild C. difficile infection, the extent of disturbance of intestinal microflora appeared to be a more important promoting factor in translocation than inflammatory activity in the mucosa. Translocation was frequent in fatal enterocolitis, with facultative species predominating in the intestinal mucosa and also C. difficile in some cases. The combination of lactobacilli and xylitol had some protective effect against C. difficile infection in these models.

Toxicity studies of amphetamine sulfate, ampicillin trihydrate, codeine, 8-methoxypsoralen, alpha-methyldopa, penicillin VK and propantheline bromide in rats and mice.

Thirteen-week toxicity studies in F344/N rats and B6C3F1 mice were conducted to determine general toxicity and target organ toxicity with amphetamine sulfate, ampicillin trihydrate, codeine, 8-methoxypsoralen, alpha-methyldopa sesquihydrate, penicillin VK, and propantheline bromide. This paper discusses the toxicity observed; use of the toxicity data to set dose levels for subsequent 2-year studies; and comparison of dose levels administered to rodents with doses used in the treatment of human disease. Drugs were administered orally in the feed or by gavage. The lowest doses in the 13-week studies were comparable to therapeutic doses in man on a mg/m2 (body surface area) basis or 5-10 times doses used in man on a mg/kg body weight basis. Little toxicity was seen at the low dose level with ampicillin, penicillin VK, 8-methoxypsoralen or propantheline bromide. At higher doses, target organ toxicity seen included the urinary bladder in male rats after propantheline bromide; the glandular stomach in rats and mice after penicillin VK; the liver and adrenals in rats after 8-methoxypsoralen; and the kidney in mice and rats after alpha-methyldopa. After amphetamine, codeine, or ampicillin administration, no target organ toxicity was seen in rats or mice, even at doses which caused body weight gain depression. The high doses chosen for subsequent 2-year studies were within 10 times human dose levels when compared on a mg/m2 body surface area.

A-16686, a new antibiotic from Actinoplanes. II. Biological properties.

A-16686, a new glycoproteide antibiotic obtained from fermentation of an Actinoplanes strain, is active against Gram-positive aerobic and anaerobic bacteria; MIC values ranged from 0.016 to 2.0 micrograms/ml. A-16686 is bactericidal for growing cells of Staphylococcus aureus, S. epidermidis, Streptococcus faecalis, S. faecium, S. mutans, S. mitis and S. sanguis. There is no cross-resistance with clinically used antibiotics. A-16686, administered subcutaneously, is very effective in experimental S. pyogenes and S. pneumoniae septicemias in the mouse.

Studies on 6-acetylmethylenepenicillanic acid (Ro 15-1903). III. Relationship between in vitro activity and chemical stability.

To resolve discrepancies between its enzymological activity and its in vitro or in vivo activity, 6-acetylmethylenepenicillanic acid (Ro 15-1903), a potent beta-lactamase inhibitor, was investigated for its chemical stability and its ability to penetrate the bacterial cell envelope. Although Ro 15-1903 was fairly stable in water or saline, it was found to be unstable in a rich medium, in mouse plasma and in human serum. Decomposition half-lives in Tryptic Soy Broth (TSB) and mouse plasma were determined by spectrometry to be 1.3 hours and 12 minutes respectively. These values were confirmed by a biochemical method for determination of Ro 15-1903. Furthermore, a large enhancement of the in vitro activity was noticed when the assay medium was changed from TSB to a synthetic medium in which Ro 15-1903 was more stable. The ampicillin-potentiating activity marginally increased if a permeability mutant harboring the R6K plasmid, which codes for TEM-1 beta-lactamase production, was used instead of the wild-type strain. These results prove that the chemical instability of Ro 15-1903 is the main cause of its disproportionally low activity in vitro and in vivo. Ro 15-1903 is not nonspecifically inactivated by proteins, since it did not lose its activity after incubation with bovine serum albumin (50 mg/ml) for 2 hours at 37 degrees C. It seems to react specifically with beta-lactamase.

Synthesis and antibacterial activity of triazole and isoxazole derivatives of ampicillin.

In order to improve the antibacterial activity of ampicillin, new penicillin derivatives having a 1-aryltriazole-4-carboxamide group or a 5-arylisoxazole-3-carboxamide group at the alpha-position of benzylpenicillin or p-hydroxybenzylpenicillin were synthesized. Some compounds in these series were found to possess high activity against Pseudomonas and other Gram-negative bacteria. In addition, structure-activity relationships, especially the effect of the hydrophobic character of the compounds on activity, were investigated.

Evaluation of antimicrobial agents for veterinary use in the ecotoxicity test using microalgae.

The influence of antimicrobial agents approved as veterinary drugs in Japan on the growth of green algae, Selenastrum capricornutum and Chlorella vulgaris, was studied in accordance with the OECD guidelines for testing chemicals. Among the agents tested, growth inhibitory activity was very varied, i.e. erythromycin showed the strongest activity (EC50, 50% effective concentration, = 0.037 mg/l), sulfa drugs had activity to some extent (EC50s of sulfamethoxazole, sulfadiazine, and sulfadimethoxine were 1.5, 2.2, and 2.3 mg/l, respectively), but ampicillin and cefazolin did not inhibit growth (EC50s>1000 mg/l). We also investigated synergistic effect of combining sulfa drugs with trimethoprim or pyrimethamine, which are commonly used as a combined drug. By adding trimethoprim, the growth inhibitory activity of sulfamethoxazole and sulfadiazine was significantly enhanced. Growth inhibition by sulfa drugs was reduced by the addition of folic acid, indicating that they inhibit folate synthesis in green algae.

Altered immune reactivity of mice born from mothers treated with ampicillin during gestation.

The cellular and humoral immune responses of 30-day-old mice born from mothers treated with ampicillin during pregnancy were studied. A lowering of cellular immunity, accompanied by a decrease in T lymphocyte percentage in lymph nodes, and an increase of humoral immune response were observed.