Humans ; Animals ; Culicidae ; Flavivirus ; Cell Line ; Spiramycin

Carrimycin (CAM) is a new antibiotics with isovalerylspiramycins (ISP) as its major components. It is produced by Streptomyces spiramyceticus integrated with a heterogenous 4″-O-isovaleryltransferase gene (ist). However, the present CAM producing strain carries two resistant gene markers, which makes it difficult for further genetic manipulation. In addition, isovalerylation of spiramycin (SP) could be of low efficiency as the ist gene is located far from the SP biosynthesis gene cluster. In this study, ist and its positive regulatory gene acyB2 were inserted into the downstream of orf54 gene neighboring to SP biosynthetic gene cluster in Streptomyces spiramyceticus 1941 by using the CRISPR-Cas9 technique. Two new markerless CAM producing strains, 54IA-1 and 54IA-2, were obtained from the homologous recombination and plasmid drop-out. Interestingly, the yield of ISP in strain 54IA-2 was much higher than that in strain 54IA-1. Quantitative real-time PCR assay showed that the ist, acyB2 and some genes associated with SP biosynthesis exhibited higher expression levels in strain 54IA-2. Subsequently, strain 54IA-2 was subjected to rifampicin (RFP) resistance selection for obtaining high-yield CAM mutants by ribosome engineering. The yield of ISP in mutants resistant to 40 μg/mL RFP increased significantly, with the highest up to 842.9 μg/mL, which was about 6 times higher than that of strain 54IA-2. Analysis of the sequences of the rpoB gene of these 7 mutants revealed that the serine at position 576 was mutated to alanine existed in each sequenced mutant. Among the mutants carrying other missense mutations, strain RFP40-6-8 which carries a mutation of glutamine (424) to leucine showed the highest yield of ISP. In conclusion, two markerless novel CAM producing strains, 54IA-1 and 54IA-2, were successfully developed by using CRISPR-Cas9 technique. Furthermore, a novel CAM high-yielding strain RFP40-6-8 was obtained through ribosome engineering. This study thus demonstrated a useful combinatory approach for improving the production of CAM.
CRISPR-Cas Systems/genetics* ; Genetic Engineering ; Ribosomes ; Spiramycin ; Streptomyces/genetics*

Ocular toxoplasmosis is the most frequent etiological factor of the known retinochoroiditis in the world. But there are very few cases of confirmed toxoplasmosis in Korea. In this paper we report a 5 year old girl who had suffered from left visual disturbance since Aug, 1978. The HA. titer for toxoplasmosis was 1:2048 and she had a typical focal exudative retinochoroiditis in the macular lesion and one small daughter lesion on superior temporal retinal arteriolar branch. She was treated with oral acetyl spiramycin(600 mg.) for 6 months. After all the active retinochoroidal lesion was changed into scar tissue and the vision has improved slightly from 0.02 to 0.2 during the followup period of one year. And also the HA. titer had decreased to 1:512. The fluorescein angiographic finding could be a helpful method to determine the healed lesion after anti toxoplasmic theraphy.
Child, Preschool ; Cicatrix ; Female ; Fluorescein ; Follow-Up Studies ; Humans ; Korea ; Nuclear Family ; Retinaldehyde ; Spiramycin* ; Toxoplasmosis ; Toxoplasmosis, Ocular*

Toxoplasma gondii, an intracellular coccidian protozoan, is the causative agent of toxoplasmosis, a widespread infection affecting various birds and mammals including humans. In immunocompetent hosts, the infection is usually asymptomatic and benign. Toxoplasmosis is either congenital or acquired. In general prenatal therapy of congenital toxoplasmosis is beneficial in reducing the ncy of infant infection. Therapies are based primarily on spiramycin because of the relative lack of toxicity and high concentration achieved in the placenta. Clindamycin is the standard drug for chemoprophylaxis in newborn infants, and is directed at preventing the occurrence of retinochoroiditis as a late sequel to congenital infection. The standard treatment for acquired toxoplasmosis in both immunocompetent and immunodeficient patients is the synergistic combination of pyrimethamine and sulphonamides. Toxoplasmic encephalitis is tbe most common manifestation of acquired toxoplasmosis in immunocompromised patients and if not treated is fatal. However, because of toxicity, the therapeutic efficacy of pyrimethamine sulphonamide combinations may be seriously limited in immunodeficient patients. We have experienced a case of toxoplasmosis during the workup of habitual aborter. So we report this case with a brief review of literatures.
Birds ; Chemoprevention ; Clindamycin ; Encephalitis ; Humans ; Immunocompromised Host ; Infant ; Infant, Newborn ; Mammals ; Placenta ; Pyrimethamine ; Spiramycin ; Toxoplasma ; Toxoplasmosis* ; Toxoplasmosis, Congenital

Macrolide antibiotics are broad-spectrum, with activity against a range of Gram-positive, Gram-negative organisms and some anaerobes. The components of macrolide antibiotics are generally complicated. Therefore, it is very important to establish impurity profiles of these antibiotics to ensure their safety and process control. Compared with classical methods, the liquid chromatography-mass spectrometry method is particularly advantageous to characterize minor components at trace levels in terms of sensitivity, efficiency and selectivity, thus more and more widely used in establishments of impurity profiles. In this study, the general approaches to characterize minor components in complex pharmaceutical matrix, fragmentation pathways of 14- and 16-membered macrolide antibiotics and the establishment of the impurity profile of acetylspiramycin were given to provide valuable enlightenments to establish the impurity profiles of pharmaceutical products.
Anti-Bacterial Agents ; analysis ; chemistry ; Chromatography, Liquid ; Drug Contamination ; Macrolides ; analysis ; chemistry ; Mass Spectrometry ; Spiramycin ; analogs & derivatives ; analysis ; chemistry

Spiramycin and midecamycin are 16-membered macrolide antibiotics with very similar chemical structures. Spiramycin has three components, namely spiramycin I, II and III. Spiramycin II and III are, respectively, the O-acetyl and propionyl derivatives at C3-hydroxyl group of spiramycin I. Midecamycin has four components, and the C3-hydroxyl group of midecamycin is all O-propionylated. The enzyme adding acyl group(s) at the C3-hydroxyl group during the biosynthesis of spiramycin and midecamycin is 3-O-acyltransferase. The 3-O-acyltransferases for spiramycin and midecamycin are also very similar, and presume to function when exchanged. To explore whether the 3-O-acyltransferase for midecamycin biosynthesis hold still the character of selective and efficient propionylation for spiramycin I at its C3-hydroxyl group, we inserted mdmB, the 3-O-acyltransferase gene from Streptomyces mycarofaciens ATCC 21454 for midecamycin biosynthesis, into a mutant strain of S. spiramyceticus F21, in which the 3-O-acyltransferase gene for spiramycin biosynthesis, sspA, was deleted; and the mdmB was integrated exactly into the chromosomal site where the sspA was deleted. We name this "hybrid" strain as SP-mdmB. HPLC analysis of the spiramycin produced by SP-mdmB showed that spiramycin I was still the major component, although the relative proportions of both spiramycin II and III increased significantly. We thus conclude that MdmB from Streptomyces mycarofaciens ATCC 21454 for midecamyicn biosynthesis do not hold the character of selective and efficient propionylation for spiramycin I within S. spiramyceticus F21, and this character is possibly limited in Streptomyces mycarofaciens ATCC 21454 for midecamycin biosynthesis.
Acylation ; Acyltransferases ; genetics ; metabolism ; Culture Media ; Genes, Bacterial ; Genetic Engineering ; methods ; Leucomycins ; biosynthesis ; Spiramycin ; biosynthesis ; Streptomyces ; enzymology ; genetics ; Substrate Specificity

There are several impurities in the spiramycin fermentation broth which leads to a lower yield and lower quality of the product. Four impurities in spiramycin broth have been simultaneously separated and identified by LC-ESI/MS. The generation of these impurities was attributed to the fluctuation of glucosylation in spiramycin biosynthesis. Nitrogen sources, ammonium in particular, were found to play an important role at the glucosylation. Aided with the information of LC-ESI/MS analysis and subsequent optimization of the culture medium, better culture medium of shake flask was designed, which leads to reduction of impurities by 22% - 88%.
Culture Media ; Fermentation ; Gas Chromatography-Mass Spectrometry ; methods ; Glycosylation ; Spectrometry, Mass, Electrospray Ionization ; methods ; Spiramycin ; biosynthesis ; chemistry ; isolation & purification

The paper is to report the establishment of an HPLC method for determination of the components of bitespiramycin and its products, and to evaluate the components profile of bitespiramycin and its related products. Liquid chromatography combined with mass spectrometry (LC-MS) was used to identify the nine major components of the reference substance of bitespiramycins. The nine components of bitespiramycins and its products have been quantified by HPLC with gradients elution methods. The content of the component of 4"-O-isovalerylspiramycin III was not less than 35%, the content of the components of 4"-O-isovalerylspiramycin (I + II + III) was not less than 60%, and the contents of the nine components of bitespiramycin were not less than 80%, separately. In addition to the components mentioned above, there also exists some other impurities, however, with lower contents. This gradient elution HPLC method reported by this paper is considered to be suitable for the quality control of bitespiramycin.
Anti-Bacterial Agents ; chemistry ; Chromatography, High Pressure Liquid ; methods ; Chromatography, Liquid ; methods ; Quality Control ; Spiramycin ; analogs & derivatives ; analysis ; chemistry ; Tandem Mass Spectrometry ; methods

4"-O-isovaleryltransferase gene (ist) was regulated by positive regulatory genes of midecamycin 4"-O-propionyltransferase gene (mpt) in Streptomyces lividans TK24. A BamH I ~8.0 kb fragment from Streptomyces mycarofaciens 1748 was proved that it contained mpt gene and linked with two positive regulatory genes, orf27 and orf28. Orf of mpt was replaced by orf of ist and linked with two regulatory genes or orf27 single, and individually cloned into the vectors pKC1139 or pWHM3 (high copy number), and then transformed into S. lividans TK24. The levels of mpt and ist expression were evaluated by the bio-tramsformation efficacy of spiramycin into 4"-O-acylspiramycins in these transformants. The results showed that 4"-O-isovalerylspiramycins could be detected only in the transformants containing the plasmids constructed with pWHM3. The efficacy of bio-transformation of the transformants containing two regulatory genes was higher than that of orf27 single. So, the positive regulatory genes system of mpt gene could enhance ist gene expression.
Acyltransferases ; genetics ; metabolism ; Bacterial Proteins ; genetics ; metabolism ; Gene Expression ; Genetic Vectors ; Plasmids ; Spiramycin ; analogs & derivatives ; biosynthesis ; Streptomyces ; enzymology ; genetics ; Streptomyces lividans ; metabolism ; Transformation, Genetic

AIMTo propose a novel polarographic method for the determination of acetylspiramycin (ASPM) is proposed.

METHODSIn 0.1 mol x L(-1) NH4Cl-NH3 x H2O (pH 8.9) buffer containing dissolved oxygen, ASPM yielded a sensitive parallel catalytic hydrogen wave with the peak potential of -1.63 V (vs SCE) by single sweep polarography.

RESULTSThe 2nd order derivative peak currents (i(p)") of the parallel catalytic hydrogen waves of ASPM showed a linear relationship with its concentrations in the range from 1.74 x 10(-3) microg x mL(-1) to 3.84 microg x mL(-1) (r = 0.9979, n=13). Its detection limit was 5.80 x l0(-4) microg x mL(-1) (3sigma) and RSD (n=13) was 1.24% at the concentration level of 0.871 microg x mL(-1).

CONCLUSIONThe proposed method could be applied to the determination of ASPM in ASPM tablets.

Anti-Bacterial Agents ; administration & dosage ; analysis ; Catalysis ; Hydrogen ; Hydrogen-Ion Concentration ; Oxygen ; chemistry ; Polarography ; methods ; Reproducibility of Results ; Spiramycin ; administration & dosage ; analogs & derivatives ; analysis ; Tablets