Construction of a novel carrimycin-producing strain by using CRISPR-Cas9 and ribosome engineering techniques / 生物工程学报

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.

Condução da toxoplasmose gestacional / Management of gestational toxoplasmosis

A toxoplasmose é uma doença proveniente do Toxoplasma gondii, um protozoário que tem os felinos como seu hospedeiro definitivo e os mamíferos e aves como seu hospedeiro intermediário. Tem um curso benigno e autolimitado quando acomete um indivíduo imunocompetente, no entanto a infecção durante a gestação acarreta até 50% de chance de toxoplasmose congênita, podendo causar danos severos ao feto. A virulência dos genótipos encontrados nas Américas Central e do Sul é a mais alta, comparada a Europa e América do Norte, tendo a doença um comportamento mais agressivo. Os estudos relatam a diminuição da infecção fetal em até 60% com o uso da espiramicina, usada ainda na profilaxia. Este artigo discute sobre a triagem materna pré-natal e sua necessidade, a profilaxia e o tratamento da infecção fetal ainda intraútero, com o objetivo de diminuir a transmissão vertical e as sequelas neonatais com suas implicações ao longo da vida.(AU)

Toxoplasmosis it is a disease originating from Toxoplasma gondii, a protozoan that has felines at as ultimate host and mammals and birds at as intermediate host. Has a benign and self-limiting course when affects immunocompetent individual, however, infection during pregnancy leads 50% chance of congenital toxoplasmosis and can cause severe damage to the fetus. The virulence of genotypes found in Central and South America is the highest compared to Europe and North America, having the disease a more aggressive behavior. Studies report a reduction in fetal infection 60% with the use spiramycin still used for prophylaxis. This article discusses prenatal maternal screening, prophylaxis and treatment of fetal infection still in utero with the objective of decreasing vertical transmission and neonatal sequelae with their lifelong implications.(AU)

TeleCondutas: toxoplasmose na gestação / TeleGuides: toxoplasmosis during pregnancy

A toxoplasmose, causada pelo protozoário Toxoplasma gondii, apresenta complicações graves quando adquirida no período gestacional. No Brasil, a incidência de toxoplasmose congênita varia entre 4 a 10 casos para cada 10 mil nascidos vivos, com apresentação clínica variável, incluindo alterações oculares (como coriorretinite), neurológicas (como encefalite, microcefalia e macrocefalia), sistêmicas (hepatomegalia, icterícia) e óbito fetal/neonatal. O risco de transmissão e a gravidade das complicações têm comportamentos inversos em relação à idade gestacional. A taxa de transmissão ao feto é 14% no primeiro trimestre e 60% no terceiro trimestre. Já a gravidade, tende a ser maior nas infecções adquiridas no começo da gestação. A taxa de transmissão varia entre 50% a 60% em mães não tratadas e 20% a 30% nas que receberam tratamento durante a gestação. Por isso, a prevenção da infecção, rastreamento e diagnóstico precoce são fundamentais para evitar as complicações da toxoplasmose congênita. Esta guia apresenta informação que orienta a conduta para casos de toxoplasmose na gestação no contexto da Atenção Primária à Saúde, incluindo: Rastreamento, Transmissão e prevenção, Manifestações clínicas, Gestação após infecção aguda, Diagnóstico na gestante, Conduta durante o pré-natal na APS, Tratamento da gestante, Diagnóstico de infecção fetal, Diagnóstico de infecção congênita, Encaminhamento para serviço especializado.

Toxoplasmosis congénita, una mirada en la actualidad del tratamiento; revisión de la literatura / Congenital toxoplasmosis, a look at contemporary treatment; literature review / Toxoplasmose congênita, um olhar atual no tratamento; revisão de literatura

Introducción. La Toxoplasmosis congénita constituye una causa significativa de morbi-mortalidad neonatal en países de bajos ingresos como Colombia. Puede originar prematuridad, secuelas patológicas y pérdida fetal. El tamizaje en las gestantes y, a su vez, un tratamiento oportuno y adecuado disminuye la transmisión vertical y sus nefastas secuelas. El objetivo es presentar evidencia científica actualizada sobre el tratamiento farmacológico de la Toxoplasmosis Congénita. Metodología. Se realizó una búsqueda no sistemática en bases de datos: Pubmed, Medline, Clinical Key y Springer. Se incluyeron artículos originales y de revisión de tema publicados desde enero de 2014 hasta abril de 2019. División de los temas tratados. se abordan la fisiopatología y clínica, el abordaje diagnóstico, alternativas de prevención y tratamiento. Conclusiones. En la actualidad la terapia farmacológica es limitada, los esquemas de manejos se basan en espiramicina o la combinación de sulfadiazina/pirimetamina y ácido folínico; estas moléculas no son del todo bien toleradas y presentan un amplio espectro de reacciones adversas secundario a sus efectos tóxicos; resulta necesario la ejecución de estudios aleatorizados para evaluar su efectividad. Cómo citar: Rueda-Paez YS, Valbuena-Ruiz L, Quintero-Pimiento N, Pinilla-Plata A, Sayago-Silva J. Toxoplasmosis congénita, una mirada en la actualidad del tratamiento; revisión de la literatura. MedUNAB. 2019;22(1):51-63. doi: 10.29375/01237047.2612

Introduction. Congenital Toxoplasmosis constitutes a significant cause of neonatal morbimortality in underdeveloped countries like Colombia. It can cause prematurity, pathological after-effects and fetal loss. Screening expectant mothers and in turn, a timely and adequate treatment, reduce vertical transmission and its devastating effects. The objective is to present up-to-date scientific evidence about the pharmacological treatment of Congenital Toxoplasmosis. Methodology. A non-systematic search of databases was conducted: Pubmed, Medline, Clinical Key and Springer. Original and topic review articles were included dating from January 2014 to April 2019. Division of topics covered. Physiopathology and clinical pathology, diagnostic approach, prevention and treatment alternatives were addressed. Conclusions. At this time, pharmacological therapy is limited, management schemes are based on spiramycin or a combination of sulfadiazine/pyrimethamine and folinic acid; these molecules are not very well tolerated and exhibit a wide spectrum of adverse reactions apart from their toxic effects, thus it is necessary to conduct randomized studies to evaluate its effectiveness. Cómo citar: Rueda-Paez YS, Valbuena-Ruiz L, Quintero-Pimiento N, Pinilla-Plata A, Sayago-Silva J. Toxoplasmosis congénita, una mirada en la actualidad del tratamiento; revisión de la literatura. MedUNAB. 2019;22(1):51-63. doi: 10.29375/01237047.2612

Introdução. A toxoplasmose congênita é uma causa significativa de morbidade e mortalidade neonatal em países de baixa renda, como a Colômbia. Pode causar prematuridade, sequelas patológicas e perda fetal. A triagem em gestantes e, por sua vez, um tratamento oportuno e adequado diminui a transmissão vertical e suas consequências desastrosas. O objetivo é apresentar evidências científicas atualizadas sobre o tratamento farmacológico da Toxoplasmose Congênita. Metodologia. Foi realizada uma revisão não sistemática nas bases de dados: Pubmed, Medline, Clinical Key e Springer. Foram incluídos tanto artigos originais, quanto revisões de tópicos publicados de janeiro de 2014 até abril de 2019. Divisão dos tópicos discutidos. foram abordadas a fisiopatologia e a clínica, a abordagem diagnóstica, alternativas para prevenção e tratamento. Conclusões. Atualmente, a terapia farmacológica é limitada, os esquemas terapéuticos baseiam-se na espiramicina ou na combinação de sulfadiazina/pirimetamina e ácido folínico; estas moléculas não são totalmente toleradas e apresentam um amplo espectro de reações adversas secundárias aos seus efeitos tóxicos. É necessário realizar estudos randomizados para avaliar sua eficácia. Cómo citar: Rueda-Paez YS, Valbuena-Ruiz L, Quintero-Pimiento N, Pinilla-Plata A, Sayago-Silva J. Toxoplasmosis congénita, una mirada en la actualidad del tratamiento; revisión de la literatura. MedUNAB. 2019;22(1):51-63. doi: 10.29375/01237047.2612

Efecto del tratamiento prenatal con espiramicina en la frecuencia de retinocoroiditis por toxoplasmosis congénita en una cohorte colombiana / Effect of antenatal spiramycin treatment on the frequency of retinochoroiditis due to congenital toxoplasmosis in a Colombian cohort

Resumen Introducción. La toxoplasmosis de la gestación es frecuente y grave. Hasta ahora no hay consenso sobre la utilidad del tratamiento para prevenir complicaciones oculares en el neonato. En la actualidad, uno de los medicamentos utilizados en las madres diagnosticadas es la espiramicina oral. Infortunadamente, en algunas mujeres gestantes no se hace el diagnóstico prenatal y, por esta u otras razones, no reciben el tratamiento. Objetivo. Describir la relación entre el tratamiento con espiramicina durante el embarazo en madres con toxoplasmosis de la gestación y la presentación de toxoplasmosis ocular en los recién nacidos. Materiales y métodos. Se llevó a cabo un estudio observacional descriptivo de serie de casos. Se evaluó una serie prospectiva de pacientes con toxoplasmosis de la gestación durante tres años de seguimiento en el Servicio de Retinología de la Clínica Universitaria Bolivariana de Medellín. Resultados. Se registraron 23 madres con diagnóstico de toxoplasmosis de la gestación. Quince de ellas (65 %) recibieron durante la gestación tratamiento con espiramicina en dosis de 3 g al día; uno de los neonatos (6,6 %) presentó toxoplasmosis ocular. De las ocho (35 %) pacientes que no recibieron tratamiento, cinco (62,5 %) tuvieron hijos con compromiso ocular por toxoplasma. La razón de momios (odds ratio, OR) del efecto protector contra dicho compromiso en los pacientes cuyas madres recibieron tratamiento fue de 0,04 (IC95% 0,00-0,67), con valor de p menor de 0,01 en la prueba exacta de Fisher. Solo se evidenció compromiso del sistema nervioso central por toxoplasmosis mediante las imágenes de tomografía o ecografía cerebral en dos (14 %) pacientes de las 14 en quienes se hicieron estos estudios. Los dos pacientes presentaron, además, compromiso ocular; ambos fueron diagnosticados en el momento del nacimiento y sus madres no habían recibido tratamiento prenatal. Conclusiones. Estos resultados evidencian que el tratamiento con espiramicina durante el embarazo en la toxoplasmosis de la gestación redujo en 96 % (IC95% 33-100 %) el riesgo relativo de presentar la enfermedad en el recién nacido.

Abstrat Introduction: Gestational toxoplasmosis is frequent and severe. There is still debate about the benefits of treatment against ocular manifestations in the newborn. Spiramycin treatment is used for this purpose, unfortunately prenatal diagnosis is sometimes delayed and pregnant women are not treated. Objective: To describe the relationship between treatment with spiramycin during pregnancy in mothers with gestational toxoplasmosis and development of ocular toxoplasmosis in newborns. Materials and methods: We conducted a descriptive study of a case series. We evaluated a prospective cohort of patients diagnosed with gestational toxoplasmosis during three years at the Retinology Service at the Clínica Universitaria Bolivariana in Medellín. Results: Gestational toxoplasmosis was found in 23 mothers; 15 (65%) were treated during pregnancy with 3 g per day of spiramycin, eight (35%) patients were untreated. In the treated group just one newborn developed ocular toxoplasmosis (6.6%), in contrast with five (62.5%) of the eight patients who did not receive treatment. These results suggest that pregnancy treatment reduces the relative risk of ocular toxoplasmosis in the newborn by 96% (95% CI: 33 - 100%). Only two (14%) of the patients who were evaluated, had nervous system involvement related to toxoplasmosis in CT scan or cerebral ultrasound. These two patients also developed ocular pathology and were diagnosed at the time of birth, so they did not received antenatal treatment. Conclusions: A protective effect was found against the ocular involvement in patients whose mother received treatment with spiramycin (OR=0.04;95% CI: 0.00-0.67), p<0.01 (Fisher's Exact Test).

The prevalence of genotypes that determine resistance to macrolides, lincosamides, and streptogramins B compared with spiramycin susceptibility among erythromycin-resistant Staphylococcus epidermidis

Coagulase-negative staphylococci, particularly Staphylococcus epidermidis, can be regarded as potential reservoirs of resistance genes for pathogenic strains, e.g., Staphylococcus aureus. The aim of this study was to assess the prevalence of different resistance phenotypes to macrolide, lincosamide, and streptogramins B (MLSB) antibiotics among erythromycin-resistant S. epidermidis, together with the evaluation of genes promoting the following different types of MLSB resistance:ermA, ermB, ermC,msrA, mphC, and linA/A’. Susceptibility to spiramycin was also examined. Among 75 erythromycin-resistantS. epidermidis isolates, the most frequent phenotypes were macrolides and streptogramins B (MSB) and constitutive MLSB (cMLSB). Moreover, all strains with the cMLSB phenotype and the majority of inducible MLSB (iMLSB) isolates were resistant to spiramycin, whereas strains with the MSB phenotype were sensitive to this antibiotic. The D-shape zone of inhibition around the clindamycin disc near the spiramycin disc was found for some spiramycin-resistant strains with the iMLSB phenotype, suggesting an induction of resistance to clindamycin by this 16-membered macrolide. The most frequently isolated gene was ermC, irrespective of the MLSB resistance phenotype, whereas the most often noted gene combination wasermC, mphC, linA/A’. The results obtained showed that the genes responsible for different mechanisms of MLSB resistance in S. epidermidis generally coexist, often without the phenotypic expression of each of them.

Spiramycin: a safe and effective option for treatment of ocular toxoplasmosis

Ocular Toxoplasmosis is a non-curable infectious disease caused by Toxoplasma Gondii and is one of the leading causes of severe visual impairment due to the involvement of posterior segment of the eye. Involvement of posterior pole causes severe visual impairment in many patients

We report a case of ocular Toxoplasmosis in a 12-year-old female child. The child was clinically diagnosed as having ocular Toxoplasmosis involving the whole posterior pole of the left eye and few parafoveal lesions in the right eye. Follow-up visit revealed new chorioretinitis lesions in the left eye. The patient was treated with oral Spiramycin 1500 mg/day in two divided doses with prednisolone tablet [Img/kg body weight] for a period of 6 weeks. The chorioretinitis lesions regressed in 3 weeks and follow-up one year after cessation of treatment revealed no recurrence. Spiramycin proved to be effective, safe, economic and easy to administer as twice daily oral dose

Expression of 4"-O-isovaleryltransferase gene from Streptomyces thermotolerans in Streptomyces lividans TK24 / 生物工程学报

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.

Impurity profiling of macrolide antibiotics by liquid chromatography-mass spectrometry / 药学学报

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.

The effect of spiramycin on Porphyromonas gingivalis and other classic periopathogens

Los estudios clínicos indican un efecto adicional beneficioso de la espiramicina cuando se la utiliza junto con el raspaje y alisado radicular. Hasta el presente, su efecto sobre la microbiota periodontal ha sido estudiado sólo con microscopio de campo oscuro. El objetivo del presente estudio fue evaluar el efecto de la espiramicina sobre Porphyromonas gingivalis y otros patógenos periodontales "clásicos", con la técnica de PCR, utilizando el gen 16S del ARNr. Participaron de este estudio clínico controlado randomizado 32 sujeros adultos, no fumadores, con enfermedad periodontal no tratada, con bolsas periodontales >- a 7mm. Se registraron parámetros clínicos en el día -15, 15, 30 y 90 de iniciado el estudio. Se analizó el biofilm periodontal subgingival con PCR, identificándose Prophyromonas gingivalis (Pg), Tannarella forsythia (Tf), Treponema denticola (Td) y Aggregatibacter actinomycetemcomitans (Aa) en el día 30 y 90. En el día 0, 25 sujetos positivos para Porphyromonas gingivalis se dividieron aleatoriamente en dos grupos: grupo Test (Espiramicina 7 días) y grupo control (Placebo 7 días). La espiramicina fue efectiva sobre Pg, Tf, Td. Hubo diferencias (p<0,05) con el grupo control. Ninguno de los grupos mostró diferencias para Aa. Estos datos indicarían que la Espiramicina fue efectiva sobre las bacterias que actualmente se asocian con periodontitis severas (Pg, Tf, Td) luego de la administración sistémica durante 7 días.

Effect of branched-chain amino acids, valine, isoleucine and leucine on the biosythesis of bitespiramycin 4"-O-acylspiramycins

Bitespiramycin, a group of 4"-O-acylated spiramycins with 4"-O-isovalerylspiramycins as the major components, was produced by recombinantspiramycin-producing strain Streptomyces spiramyceticus harboring a 4"-O-acyltransferase gene. The experiment was initially performed in synthetic medium with 0.5 g l-1 Valine, Isoleucine or Leucine feeding at 36 h cultivation. When valine was fed, the biological titer of bitespiramycin was 45.3 percent higher than that of the control group, but the relative content of total isovalerylspiramycin components decreased by 22.5 percent. In the case of ilecine, the biological titer of bitespiramycin and the total isovalerylspiramycins alone were 85 percent and 72.1 percent of the control group, respectively. In contrast, the relative content of other acylated spiramycins increased by 54.41 percent. However, leucine feeding increased the relative content of total isovalerylspiramycins by 41.9 percent while the biological titer of bitespiramycin was nearly equal to that of the control group. The improvement effect of leucine on the biosynthesis of isovalerylspiramycins was further confirmed by feeding of 2.0 g l-1 leucine to the culture with complex medium. After batch feeding with a total amount of 2.0 g l-1 leucine to the culture from 70 h to 90 h, the biological titer of bitespiramycin was almost unreduced, and the final relative content of total isovalerylspiramycins increased from 31.1 percent to 46.9 percent.

Options for clinical trials of pre and post-natal treatments for congenital toxoplasmosis

Clinical trials comparing different drug regimens and strategies for the treatment of congenital toxoplasmosis and its clinical manifestations in the liveborn child in different clinical settings should aim at formally evaluating the net benefit of existing treatments and at developing new therapeutic options. Currently, there is no ideal drug for congenital toxoplasmosis; future research should focus on the screening of new active drugs and on their pre-clinical and early clinical development, with a focus on pharmacokinetic/dynamic studies and teratogenicity. For the prenatal treatment of congenital toxoplasmosis, a trial comparing spiramycine to pyrimethamine-sulphadiazine and placebo would allow a formal estimation of the effect of both drugs in infected pregnant women. In newborn children, the net benefit of pyrimethamine-sulphadiazine should also be formally assessed. These trials will be implemented in settings where prenatal screening for Toxoplasma gondii is currently implemented. Trials should be carefully designed to allow for translation to other settings and modelling tools like cost-effectiveness analysis should be used to provide clinicians and founders with the best available evidence to establish recommendations.

Diagnosis of congenital toxoplasmosis: prenatal and neonatal evaluation of methods used in Toulouse University Hospital and incidence of congenital toxoplasmosis

The aim of this study was to determine the incidence of congenital toxoplasmosis (CT) and to assess the performances of prenatal and neonatal diagnoses. From 1994-2005, in Toulouse University Hospital, France, amniocentesis was performed on 352 pregnant women who were infected during pregnancy. All women were treated with spiramycin and pyrimethamine-sulfadoxine when prenatal diagnosis was positive. Among the 275 foetuses with follow-up, 66 (24 percent) were infected. The transmission rates of Toxoplasma gondii were 7 percent, 24 percent and 59 percent in the first, second and third trimesters, respectively. The sensitivity and specificity of PCR on amniotic fluid (AF) were 91 percent and 99.5 percent, respectively. One case was diagnosed by mouse inoculation with AF and six cases were diagnosed by neonatal or postnatal screening. The sensitivity and specificity of PCR on placentas were 52 percent and 99 percent, respectively. The sensitivity of tests for the detection of specific IgA and IgM in cord blood was 53 percent and 64 percent, respectively, and specificity values were 91 percent and 92 percent. In conclusion, PCR performed on AF had the highest levels of sensitivity and specificity for the diagnosis of CT. This permits an early diagnosis of most cases and should be recommended.

Determination of the components of bitespiramycin by HPLC / 药学学报

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.

Acylation specificity of midecamycin 3-O-acyltransferase within Streptomyces spiramyceticus F21 / 生物工程学报

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.

Papel da Criptosporidiose na doença inflamatória intestinal / The role of cryptosporidiosis in inflammatory bowel disease

Relata-se o caso de um paciente com quadro diarréico sem antecedente de doença intestinal prévia que, inicialmente, foi tratado para a infecção por Cryptosporidium sp. Na evolução, mostrou ser portador de doença inflamatória intestinal

New effective and safe combination therapy for acute and chronic toxoplasmosis in murine models

Toxoplasma gondii is a protozoan parasite that infects humans and most species of warm blood animals. The most effective treatment for toxoplasmosis is the classic combination of pyrimethamine/sulfadiazine while the safest drug is spiramycin. These traditional anti-Toxoplasma drugs are either ineffective or have serious side effects that sometimes needs discontinuation of treatment. Both mouse [acute] and rat [chronic] models were used to evaluate a novel dipyridamole/allopurinol anti-Toxoplasma combination therapy that targets the purine salvage pathways of the parasite. The efficacy and safety of the new drugs were evaluated in comparison with traditional therapies; pyrimethamine/sulfadiazine and spiramycin. The life expectancy of mice in dipyridamole/ allopurinol group was significantly increased in comparison to other drug groups and almost doubled in relation to the infection control group. A significant reduction of anti-Toxoplasma antibody titers was only present in dipyridamole/allopurinol group in comparison to the infection control groups in both acute and chronic states of infection. The drug proves to be safe as evidenced by normal blood parameters reflecting no sign of drug toxicity. Pyrimethamine/sulfadiazine combination was second in efficacy while spiramycin was second in safety

LC-ESI/MS identification and reduction of impurities in spiramycin fermentation / 生物工程学报

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%.

Parallel catalytic hydrogen wave of acetylspiramycin caused by dissolved oxygen and its application / 药学学报

<p><b>AIM</b>To propose a novel polarographic method for the determination of acetylspiramycin (ASPM) is proposed.</p><p><b>METHODS</b>In 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.</p><p><b>RESULTS</b>The 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).</p><p><b>CONCLUSION</b>The proposed method could be applied to the determination of ASPM in ASPM tablets.</p>