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.

iTRAQ-based quantitative proteomic analysis of Microcystis aeruginosa exposed to spiramycin at different nutrient levels.

Research on the combined effects of antibiotic contaminants and environmental factors in cyanobacteria is still limited. This study focused on the action and its mechanism of spiramycin combined with changes in nitrogen and phosphorus level in Microcystis aeruginosa at environmentally relevant concentrations. Though photosynthetic activity was stimulated by spiramycin at a high nutrient level, no significant correlation (p>0.05) was found between photosynthesis-related proteins and growth-related proteins, and the growth rate was inhibited by 200ngL-1 of spiramycin. At low nitrogen and low phosphorus levels, up-regulated photosynthesis-related proteins were closely correlated with (p<0.05) stress response-related, transcription-related and cell division-related proteins, which consequently led to stimulated growth of M. aeruginosa under spiramycin exposure. Spiramycin exposure also regulated the production of microcystins (MCs) and the expression of two microcystin synthetases (mcyB and mcyC). The spiramycin-induced protein secretion process and the up-regulation of ATP binding cassette transporters might contribute to the increased MC release. Enolase, superoxide dismutase, protein GrpE, DNA-directed RNA polymerase subunit alpha and serine protease were candidate target proteins of spiramycin in M. aeruginosa under different nutrient conditions. Coexisting spiramycin mitigated the threat of cyanobacteria to aquatic environments at a high nutrient level but aggravated cyanobacterial bloom at a low nitrogen level.

Antiparasitic effects of oxymatrine and matrine against Toxoplasma gondii in vitro and in vivo.

Toxoplasma gondii (T. gondii) is an important pathogen which can causes serious public health problems. Since the current therapeutic drugs for toxoplasmosis present serious host toxicity, research on effective and new substances of relatively low toxicity is urgently needed. This study was carried out to evaluate the anti-parasitic effect of oxymatrine (OM) and matrine (ME) against T. gondii in vitro and in vivo. In our study, the anti-T. gondii activities of ME and OM were evaluated in vitro using cell counting kit-8 assay, morphological observation and trypan blue exclusion assay. In vivo, mice were sacrificed four days post-infection and ascites were drawn out to determine the extent of tachyzoite proliferation. Viscera indexes and liver biochemical parameters, such as alanine aminotransferase (ALT), aspartate aminotransferase (AST), glutathione (GSH) and malondialdehyde (MDA), were examined to evaluate the toxicity of compounds to mice. As a result, OM and ME showed anti-T. gondii activity but low selectivity toxicity to HeLa cells. Both compounds also significantly decreased the number of tachyzoites in peritoneal cavity and recovered the levels of ALT, AST, GSH and MDA in liver. Moreover, the mice treated with OM or ME achieved better results in viscera index and survival rate than that of spiramycin. These results suggest that OM and ME are likely the sources of new drugs for toxoplasmosis, and further studies will be necessary to compare the efficacy of drug combination, as well as identify its action of mechanism.

Combined effects of two antibiotic contaminants on Microcystis aeruginosa.

Combined toxicity of spiramycin and amoxicillin was tested in Microcystis aeruginosa. The respective 50% effective concentrations (EC50mix) expressed in toxic unit (TU) values were 1.25 and 1.83 for spiramycin and amoxicillin mixed at 1:7 and 1:1, suggesting an antagonistic interaction at the median effect level. Deviations from the prediction of concentration addition (CA) and independent action (IA) models further indicated that combined toxicity of two antibiotics mixed at 1:1 varied from synergism to antagonism with increasing test concentration. Both the EC50mix of 0.86 (in TU value) and the deviation from two models manifested a synergistic interaction between spiramycin and amoxicillin mixed at 7:1. At an environmentally relevant concentration of 800ngL(-1), combined effect of mixed antibiotics on algal growth changed from stimulation to inhibition with the increasing proportion of higher toxic component (spiramycin). Chlorophyll-a content and expression levels of psbA, psaB, and rbcL varied in a similar manner as growth rate, suggesting a correlation between algal growth and photosynthesis under exposure to mixed antibiotics. The stimulation of microcystin-production by mixed antibiotics was related with the elevated expression of mcyB. The mixture of two target antibiotics with low proportion of spiramycin (<50%) could increase the harm of M. aeruginosa to aquatic environments by stimulating algal growth and production and release of microcystin-LR at their current contamination levels.

Influences of two antibiotic contaminants on the production, release and toxicity of microcystins.

The influences of spiramycin and amoxicillin on the algal growth, production and release of target microcystins (MCs), MC-LR, MC-RR and MC-YR, in Microcystis aeruginosa were investigated through the seven-day exposure test. Spiramycin were more toxic to M. aeruginosa than amoxicillin according to their 50 percent effective concentrations (EC(50)) in algal growth, which were 1.15 and 8.03 µg/l, respectively. At environmentally relevant concentrations of 100 ng/l-1 µg/l, spiramycin reduced the total MC content per algal cell and inhibited the algal growth, while exposure to amoxicillin led to increases in the total MC content per algal cell and the percentage of extracellular MCs, without affecting the algal growth. Toxicity of MCs in combination with each antibiotic was assessed in the luminescent bacteria test using the toxic unit (TU) approach. The 50 percent effective concentrations for the mixtures (EC(50mix)) were 0.56 TU and 0.48 TU for MCs in combination with spiramycin and amoxicillin, respectively, indicating a synergistic interaction between MCs and each antibiotic (EC(50mix)<1TU). After seven-day exposure to 100 ng/l-1 µg/l of antibiotics, spiramycin-treated algal media and amoxicillin-treated algal media showed significantly lower (p<0.05) and higher (p<0.05) inhibition on the luminescence of Photobacterium phosphoreum, respectively, compared with the untreated algal medium. These results indicated that the toxicity of MCs were alleviated by spiramycin and enhanced by amoxicillin, and the latter effect would increase threats to the aquatic environment.

Development and validation of a potentiometric biosensor assay for tylosin with demonstrated applicability for the detection of two other antimicrobial growth-promoter compounds in feedstuffs.

A potentiometric biosensor assay based on a commercially available polyclonal antibody was developed to detect tylosin residues in animal feed. The method can be used as a rapid (less than 45 min) laboratory-based procedure or as a portable field-test for the simultaneous measurement of up to 12 different samples. For both procedures the qualitative detection capability (CCß) for tylosin was determined as 0.2 mg kg(-1) in a range of animal feeds with a measurement repeatability at concentrations between 0.2 and 4 mg kg(-1) of ≤13% coefficient of variation (%CV). The field-test format was capable of detecting tylosin residues at operating (external air) temperatures ranging between +4 and 37°C, although some reduction in signal was observed at the lower temperatures. The laboratory-based tylosin assay was evaluated using 16 medicated and 22 non-medicated feeds and was found to give comparable data with a confirmatory method based upon liquid chromatography-tandem mass spectrometry (LC-MS/MS). The potential to develop a multi-probe format assay for the simultaneous detection of tylosin, spiramycin and virginiamycin was also demonstrated. Cross-validation in a second laboratory showed the assay to be transferable, reliable and robust.

Algal toxicity of antibacterial agents used in intensive farming.

The growth inhibiting effects of eight antibiotics used either therapeutically or as growth promoters in intensive farming on two species of micro algae, Microcystis aeruginosa (freshwater cyanobacteria) and Selenastrum capricornutum (green algae) were investigated. The effects of the antibiotics benzylpenicillin (penicillin G) (BP), chlortetracycline (CTC), olaquindox (O), spiramycin (SP), streptomycin (ST), tetracycline (TC), tiamulin (TI) and tylosin (TY) were tested in accordance with the ISO 8692 (1989) standard protocol. Algal growth was measured as increase in chlorophyll concentration by extraction with ethanol followed by measurement of fluorescence. Results were quantified in terms of growth rates using the Weibull equation to describe the concentration response relationship. The toxicity (EC50 value, mg/l) in alphabetic order were BP (0.006); CTC (0.05); O (5.1); SP (0.005); ST (0.007); TC (0.09); TI (0.003) and TY (0.034) for M. aeruginosa. BP (NOEC = 100); CTC (3.1); O (40); SP (2.3); ST (0.133); TC (2.2); TI (0.165) and TY (1.38) for S. capricornutum. In this investigation M. aeruginosa is found to be about two orders of magnitude more sensitive than S. capricornutum. It was observed that most of the compounds were unstable during the test period due to hydrolysis and photolysis.

[Studies on acetylspiramycin. II. Biological activities of spiramycin components].

Acetylspiramycin (ASPM) was fractionated using high performance liquid chromatography (HPLC). The peak fractions were named F1 to F7 successively in order of increasing retention times (Rt), i.e., increasing hydrophobicity, and studied for 1) antibacterial activities (MIC), 2) antibacterial potency against Bacillus subtilis ATCC 6633, 3) therapeutic effect on mice infected with Streptococcus pneumoniae III, Staphylococcus aureus Smith, 4) acute toxicity by i.p. administration to mice (LD50) and 5) cytotoxicities to fibroblasts derived from Chinese-hamster lung (CHL), cow pulmonary artery endothelial cells (CPAE) and rat hepatic cells. The results obtained are summarized below. 1. Components F1 and 4'-acetylspiramycin F2 had significantly different biological activities from those of other components: F1 showed the lowest antibacterial potency of 492 micrograms (potency)/mg, F2 showed the highest antibacterial potency of 2,040 micrograms (potency)/mg and correspondingly the lowest LD50 value of 692 mg/kg (the highest toxicity). The therapeutic effect of F2 on infections in mice was found to be the second smallest and was superior only to that of F1. The LD50 value of F1 was 1,200 mg/kg and similar to that of ASPM. 2. Antibacterial potencies of F3, F4, F5 and F6 were 1,165, 1,266, 1,374 and 1,530 micrograms (potency)/mg, respectively; fraction with the higher antibacterial activities corresponded to the longer retention times, i.e., the greater hydrophobicities. The most hydrophobic component, F7, 3-propionyl-3",4"-diacetylspiramycin, however, showed a low antibacterial potency of 1,085 micrograms (potency)/mg, next to the lowest one, F1, a fact which was in contradiction to with the sequential relation between hydrophobicities and potencies from F3 to F6.(ABSTRACT TRUNCATED AT 250 WORDS)