Characterization of two malaria parasite organelle translation elongation factor G proteins: the likely targets of the anti-malarial fusidic acid

Malaria parasites harbour two organelles with bacteria-like metabolic processes that are the targets of many anti-bacterial drugs. One such drug is fusidic acid, which inhibits the translation component elongation factor G. The response of P. falciparum to fusidic acid was characterised using extended SYBR-Green based drug trials. This revealed that fusidic acid kills in vitro cultured P. falciparum parasites by immediately blocking parasite development. Two bacterial-type protein translation elongation factor G genes are identified as likely targets of fusidic acid. Sequence analysis suggests that these proteins function in the mitochondria and apicoplast and both should be sensitive to fusidic acid. Microscopic examination of protein-reporter fusions confirm the prediction that one elongation factor G is a component of parasite mitochondria whereas the second is a component of the relict plastid or apicoplast. The presence of two putative targets for a single inhibitory compound emphasizes the potential of elongation factor G as a drug target in malaria.     

Morphological stages of Bacillus subtilis sporulation and resistance to fusidic acid.

During spore development of Bacillus subtilis both protein synthesis and sporulation become resistant to the antibiotic fusidic acid. This resistance develops at the time when asymmetric prespore septa are formed. Simultaneously ribosomes lose their ability to bind fusidic acid, as demonstrated by their affinity chromatography with the immobilized drug. Mutants resistant to fusidic acid during growth are oligosporogenous; their sporulation development is blocked before septum formation. These results indicate that normal ribosomes are needed for prespore septation sporulation; only after septation can protein synthesis be maintained, throughout the development period, by fusidate resistant ribosomes.     

Staphylococcal bacteraemia,fusidic acid,and jaundice.

Fusidic acid was used to treat 131 out of 250 patients with staphylococcal bacteraemia over 10 years. Other antimicrobial agents were given to the 119 remaining patients. Thirty-seven patients were already jaundiced before antibiotic treatment was started. Jaundice developed during treatment in 38 out of 112 patients given fusidic acid (34%) and in two out of 101 patients given other antimicrobials. The incidence of jaundice was higher in patients given fusidic acid intravenously (48%) rather than by mouth (13%). Jaundice appeared within 48 hours after the administration of fusidic acid in 93% of these cases. When the drug was stopped serum bilirubin concentrations fell to normal values within four days in those patients in whom they had been previously normal and who survived the bacteraemic episode. Fusidic acid was associated with increasing jaundice in 13 of 19 patients (68%) already jaundiced before it was given. In six out of 32 patients who developed jaundice while receiving intravenous fusidic acid serum alkaline phosphatase activity was raised suggestive of cholestatic jaundice. The mechanism in the remaining patients was unknown. Fusidic acid, particularly the intravenous preparation, in invaluable in treating severe staphylococcal infection but should be used with caution in patients with abnormal liver function. Patients receiving intravenous fusidic acid should be given the oral form of the drug as soon as their clinical condition permits.     

Fusidic-acid-resistant staphylococci

Penicillin-resistant staphylococci were examined to determine the spontaneous mutation frequency of resistance to antistaphylococcal antibiotics including fusidic acid, cephalothin and lincomycin. Fusidic-acid-resistant mutants were obtained about twenty times more frequently than cephalothin-resistant mutants and about one hundred times more frequently than lincomycin-resistant mutants. Among the fusidic-acid-resistant mutants, ninety-seven per cent were resistant to 20 g/ml and thirty percent to 100 g/ml of the drug. The fusidic-acid-resistant mutants had prolonged generation times not related to the level of drug resistance. Cross-resistance between fusidic acid and cephalothin and between lincomycin and erythromycin was detected. Preliminary evidence was presented indicating that fusidic-acid-resistant mutants inactivate fusidic acid to a greater extent than sensitive cells.     

Antibiotic treatment of abscesses of the central nervous system.

Samples of intracranial pus and serum from 32 patients were assayed to determine the concentrations reached in them of penicillin, ampicillin, cloxacillin, cephaloridine, gentamicin, chloramphenicol, fusidic acid, and lincomycin. Metronidazole had not been given. Penicillin penetrated abscesses reasonably well, but other beta-lactam antibiotics did not. The penetration of chloramphenicol was erratic. Aminoglycosides penetrated poorly, but lincomycin and fusidic acid penetrated well. Assay of sulphonamides and co-trimoxazole in pus was unreliable. These studies indicate that treatment of abscesses of the central nervous system should be considered according to the site and the likely antecedent cause. Abscesses of sinusitic origin, usually in the frontal lobe, yield penicillin-sensitive streptococci. Penicillin is the drug of choice. Abscesses of otitic origin, usually in the temporal lobe, yield a mixed flora, often including anaerobic bacteria. Multiple antibiotic therapy is indicated. Abscesses of metastatic or cryptogenic origin yield streptococci or mixed cultures, and multiple therapy is appropriate while awaiting the bacteriological results. Spinal and post-traumatic abscesses yield Staphylococcus aureus, and fusidic acid is the drug of choice.     

Molecular analysis of fusidic acid resistance in Staphylococcus aureus

Fusidic acid is a potent antibiotic against severe Gram-positive infections that interferes with the function of elongation factor G (EF-G), thereby leading to the inhibition of bacterial protein synthesis. In this study, we demonstrate that fusidic acid resistance in Staphylococcus aureus results from point mutations within the chromosomal fusA gene encoding EF-G. Sequence analysis of fusA revealed mutational changes that cause amino acid substitutions in 10 fusidic acid-resistant clinical S. aureus strains as well as in 10 fusidic acid-resistant S. aureus mutants isolated under fusidic acid selective pressure in vitro. Fourteen different amino acid exchanges were identified that were restricted to 13 amino acid residues within EF-G. To confirm the importance of observed amino acid exchanges in EF-G for the generation of fusidic acid resistance in S. aureus, three mutant fusA alleles encoding EF-G derivatives with the exchanges P406L, H457Y and L461K were constructed by site-directed mutagenesis. In each case, introduction of the mutant fusA alleles on plasmids into the fusidic acid-susceptible S. aureus strain RN4220 caused a fusidic acid-resistant phenotype. The elevated minimal inhibitory concentrations of fusidic acid determined for the recombinant bacteria were analogous to those observed for the fusidic acid-resistant clinical S. aureus isolates and the in vitro mutants containing the same chromosomal mutations. Thus, the data presented provide evidence for the crucial importance of individual amino acid exchanges within EF-G for the generation of fusidic acid resistance in S. aureus.     

Fusidic acid: new opportunities with an old antibiotic.

The extensive foreign experience with fusidic acid prior to its belated introduction to Canada is reviewed. Fusidic acid is a steroid antibiotic with minimal toxic and hormonal effects that is mainly excreted through the liver. It has a predominantly bactericidal action and does not shown cross-resistance with other antibiotics. Since organisms resistant to this drug form easily in vitro when exposed to low concentrations, complementary treatment with another antibiotic may be required in some clinical situations. Although fusidic acid is active in vitro against a number of organisms, to date it has mainly been used to treat serious infections due to Staphylococcus aureus. The agent appears to be particularly valuable in the treatment of bone and joint infections and in pediatric practice. Fusidic acid will soon be available in Canada for both oral and intravenous administration. Attainable antibiotic levels in many tissues and body fluids greatly exceed the minimum inhibitory concentrations.     

A spectrophotometric method of analysis of fusidic acid

A spectrophotometric method for assay of fusidic acid is described. The method is based on reaction with a reagent consisting of acetic anhydride and concentrated sulfuric acid. Mathematical processing of the results of the main substance determination in fusidic acid preparations showed that the error did not exceed 2 per cent. Procedures for spectrophotometric assay of fusidic acid in control of the processes of its biosynthesis, isolation and purification were developed. The procedures provided control of the technological process of fusidic acid production.     

Pharmacokinetics of fusidic acid in laboratory animals.

The ability of evaluate the efficacy of fusidic acid in animal models of infectious disease is limited by the absence of pharmacokinetic data for the agent in laboratory animals. In our study, aspects of fusidic acid pharmacokinetics were compared in rats (Rattus norwegicus), mice (Mus musculus), rabbits (Oryctolagus cuniculus), and guinea pigs (Cavia porcellus). Sodium fusidate was poorly absorbed after oral administration to rats, although limited absorption occurred in guinea pigs, mice, and rabbits. Subcutaneous injections of diethanolamine fusidate to laboratory rats, however, achieved a serum profile similar to that observed in humans. There was no evidence of drug accumulation in rats given repeated subcutaneous doses of diethanolamine fusidate during a 4-day period, but rabbits showed clear evidence of a cumulative effect.     

Technique to Improve the Rate of Healing of Incised Abscesses

In a comparative investigation incised skin abscesses were treated by either introducing sterile fusidic acid gel into the cavity on one occasion only or applying daily superficial dressings impregnated with sodium fusidate ointment. In comparison with the dressing group, the intracavity use of fusidic acid gel reduced the mean healing time of incised abscesses by approximately one-half. When abscesses were analysed according to site and size, the reduction in mean healing time was equally striking. No hypersentisivity or irritation to fusidic acid or its sodium salt applied by either method was observed.The procedure of introducing fusidic acid gel into an incised abscess cavity is a promising alternative to superficial antibiotic dressings or wicks in the treatment of incised abscesses.     

Isolation and characterization of fusidic acid-resistant, sporulation-defective mutants of Bacillus subtilis.

Fusidic acid-resistant, sporulation-defective mutants were isolated from Bacillus subtilis 168 thy trp. About two-thirds of the fusidic acid-resistant (fusr) mutants were defective in sporulation ability and fell into three classes with respect to sporulation character. The representative mutants FUS426 and FUS429 were characterized in detail. FUS426 [fusr spo (Ts)], a temperature-sensitive sporulation mutant, grew well at 30 and 42 degrees C but did not sporulate at 42 degrees C. FUS429 [fusr spo (Con)], conditional sporulation mutant, grew and sporulated normally in the absence of fusidic acid, but its sporulation and growth rates decreased in the presence of fusidic acid, depending on the concentration of the drug. Although electron microscopic observation showed that both mutants were blocked at stage I of sporulation, the physiological analyses indicate that these mutants belong to the SpoOB class. Both mutants formed a thickened cell wall as compared with that of the parental strain. Genetic and in vitro protein synthesis analyses led to the conclusion that the sporulation-defective character of mutants FUS426 and FUS429 resulted from an alteration in elongation factor G caused by a single lesion in the fus locus. The possible role of elongation factor G in sporulation is discussed.     

Purification and characterization of a novel extracellular Streptomyces lividans 66 enzyme inactivating fusidic acid.

The wild-type strain Streptomyces lividans 66 is resistant against the steroid-like antibiotic fusidic acid. Comparative studies of the wild-type strain and a fusidic acid-sensitive mutant allowed the identification of an extracellular enzyme which inactivates fusidic acid. With the help of a combination of ultrafiltration and chromatographies with Phenyl-Sepharose and an anion exchanger, the enzyme was highly purified. Its apparent molecular mass is 48 kDa, its optimal activity ranges between 45 and 55 degrees C, and its optimal pH is 6.0 to 9.0. It is stimulated by neither monovalent nor divalent ions. The enzyme acts as a specific esterase which removes the acetyl group at C-16 from fusidic acid. The resulting intermediate is unstable, and spontaneous lactonization between C-21 and C-16 occurs rapidly.     

Mapping the fucidin resistance marker in S. typhimurium and a study of the properties of its transductants]

The fusidic resistance marker in S. typhimurium is contransduced in 95% of cases by means of P 22 phage with the streptomycin resistance marker. The transfer of the fusidic acid resistance gene doesn't lead to any significant alterations in the tranductants' properties (morphology, antigenic structure, growth rate, biochemical activity, sensitivity to other antibiotics). The fusidic acid resistant mutants and transductants studied displayed a significantly decreased virulence to albino mice. The rate of this decrease, however, doesn't correspond to the degree of transductants' resistance. Virulent variants are also possible.     

Susceptibility to and resistance determinants of fusidic acid in Staphylococcus aureus isolated from Chinese children with skin and soft tissue infections

This study aims to determine the resistance rates and determinants of fusidic acid among Staphylococcus aureus isolates collected from Chinese pediatric patients with skin and soft tissue infections (SSTIs). Between 2008 and 2009, a total of 186 clinical S. aureus isolates were collected from the pediatric patients with SSTIs, abscess (44.6%) was the most common SSTI in children 0-16 years old. Four clinical isolates (4/186, 2.2%) were resistant to fusidic acid. Two of these isolates were methicillin-resistant S. aureus (MRSA) that carry the fusC gene. The other two isolates were methicillin-sensitive S. aureus (MSSA) that carry the fusB gene. In the two fusB-positive clinical isolates, the fusB gene was located in a transposon-like element that has 99% identity with a pUB101 fragment from S. aureus. The four fusidic acid-resistant clinical isolates were ST1-MRSA-SCCmecV-t127, ST93-MRSA-SCCmecIII-t202, ST680-MSSA-t5415, and ST680-MSSA-t377. The fusidic acid resistance rate of S. aureus isolated from Chinese pediatric patients with SSTIs was low, and the genes fusB and fusC were the main resistance determinants. The transposon-like element that contains the fusB gene might participate in the transmission of fusidic acid resistance genes. This is the first report regarding the emergence of fusidic acid-resistant clinical S. aureus isolates in mainland China.     

Unique antibiotic sensitivity of archaebacterial polypeptide elongation factors.

The antibiotic sensitivity of the archaebacterial factors catalyzing the binding of aminoacyl-tRNA to ribosomes (elongation factor Tu [EF-Tu] for eubacteria and elongation factor 1 [EF1] for eucaryotes) and the translocation of peptidyl-tRNA (elongation factor G [EF-G] for eubacteria and elongation factor 2 [EF2] for eucaryotes) was investigated by using two EF-Tu and EF1 [EF-Tu(EF1)]-targeted drugs, kirromycin and pulvomycin, and the EF-G and EF2 [EF-G(EF2)]-targeted drug fusidic acid. The interaction of the inhibitors with the target factors was monitored by using polyphenylalanine-synthesizing cell-free systems. A survey of methanogenic, halophilic, and sulfur-dependent archaebacteria showed that elongation factors of organisms belonging to the methanogenic-halophilic and sulfur-dependent branches of the "third kingdom" exhibit different antibiotic sensitivity spectra. Namely, the methanobacterial-halobacterial EF-Tu(EF1)-equivalent protein was found to be sensitive to pulvomycin but insensitive to kirromycin, whereas the methanobacterial-halobacterial EF-G(EF2)-equivalent protein was found to be sensitive to fusidic acid. By contrast, sulfur-dependent thermophiles were unaffected by all three antibiotics, with two exceptions; Thermococcus celer, whose EF-Tu(EF1)-equivalent factor was blocked by pulvomycin, and Thermoproteus tenax, whose EF-G(EF2)-equivalent factor was sensitive to fusidic acid. On the whole, the results revealed a remarkable intralineage heterogeneity of elongation factors not encountered within each of the two reference (eubacterial and eucaryotic) kingdoms.     

Mutations in the G-domain of elongation factor G from Thermus thermophilus affect both its interaction with GTP and fusidic acid

Two hypersensitive and two resistant variants of elongation factor-G (EF-G) toward fusidic acid are studied in comparison with the wild type factor. All mutated proteins are active in a cell-free translation system and ribosome-dependent GTP hydrolysis. The EF-G variants with the Thr-84-->Ala or Asp-109-->Lys mutations bring about a strong resistance of EF-G to the antibiotic, whereas the EF-Gs with substitutions Gly-16-->Val or Glu-119-->Lys are the first examples of fusidic acid-hypersensitive factors. A correlation between fusidic acid resistance of EF-G mutants and their affinity to GTP are revealed in this study, although their interactions with GDP are not changed. Thus, fusidic acid-hypersensitive mutants have the high affinity to an uncleavable GTP analog, but the association of resistant mutants with GTP is decreased. The effects of either fusidic acid-sensitive or resistant mutations can be explained by the conformational changes in the EF-G molecule, which influence its GTP-binding center. The results presented in this paper indicate that fusidic acid-sensitive mutant factors have a conformation favorable for GTP binding and subsequent interaction with the ribosomes.     

Sites of action of fusidic acid in eukaryotes. Inhibition by fusidic acid of a ribosome-independent GTPase from Artemia salina embryos.

1. A ribosome-independent GTPase activity has been isolated from the high-speed supernatant fraction of Artemia salina embryos, and some of its properties have been studied. This activity is inhibited by fusidic acid, an antibiotic generally thought to inhibit only EF-2 in eukaryotes. However, several lines of evidence indicate that the GTPase activity, described here, is distinct from EF-2. The results suggest, therefore, that the inhibitory effect of fusidic acid in eukaryotic systems is not restricted to EF-2 (and ribosome)-dependent functions only. 2. The results of other experiments have revealed that, despite its ability to inhibit the GTPase activity mentioned above, fusidic acid is not a non-specific inhibitor of all ribosome-independent GTPase and ATPase activities present in eukaryotic cells.     

Molecular basis of fusB-mediated resistance to fusidic acid in Staphylococcus aureus

The primary mechanism of fusidic acid resistance in clinical strains of Staphylococcus aureus involves acquisition of the fusB determinant. The genetic elements(s) responsible are incompletely defined, and the mechanism of resistance is unknown. Here we report the cloning, sequencing and overexpression of a single gene (fusB) from plasmid pUB101 capable of conferring resistance to fusidic acid in S. aureus. The fusB gene is located on a transposon-like element and encodes a small (25 kDa), cytoplasmic protein for which homologues exist in a number of clinically important and environmental Gram-positive bacterial species. Bioinformatic analysis of regions immediately upstream of fusB suggested that expression of resistance is regulated by translational attenuation, which was confirmed through use of reporter fusions. FusB was overexpressed in Escherichia coli as a polyhistidine-tagged fusion product, and the purified protein shown to protect an in vitro staphylococcal translation system from inhibition by fusidic acid in a specific and dose-dependent fashion. Purified FusB bound staphylococcal EF-G, the target of fusidic acid. The protein provided no protection from inhibition by fusidic acid when added to an in vitro E. coli translation system, consistent both with the observed failure of FusB to bind E. coli EF-G, and its inability to confer resistance in E. coli.     

1272例尿液标本细菌培养结果及耐药性分析

目的调查临床送检中段尿标本菌群分布及体外抗生素耐药性,为临床合理使用抗生素提供依据。方法回顾性分析2010年1月至2012年6月住院患者送检中段尿标本中,分离的1272株细菌（剔除重复菌株）临床分布及耐药性。采用美国BDphoenix100全自动细菌鉴定药敏分析仪进行菌种鉴定及药敏试验,结合相关临床资料,应用WHONET5．6分析软件进行耐药性分析。结果从分离到的l272株细菌中,检出前5位的细菌是大肠埃希菌、肺炎克雷伯菌、屎肠球菌、粪肠球菌和铜绿假单胞菌,构成比分别为20．3％、14．4％、14．2％、9．4％和7．4％。肠杆菌对氨苄西林的耐药率最高为93．9％,耐药率较低的有亚胺培南、美洛培南、头孢哌酮／舒巴坦和多粘菌素B,它们的耐药率在10．5％～15．9％。非发酵菌对氯霉素、氨苄西林、头孢唑啉和阿莫西林／克拉维酸的耐药率最高在90．1％～100％,对多粘菌素B和头孢哌酮／舒巴坦的耐药率较低,分别为7．7％和16．9％。肠球菌对利奈唑胺、万古霉素、替考拉宁和呋西地酸的耐药率最低,分别为2．6％、7．2％、8．8％和8．8％;葡萄球菌对万古霉素的耐药率为0,对喹奴普汀-达福普汀的耐药率为0．8％,对呋哺妥因、替考拉宁、夫西地酸、利奈唑胺、利福平的耐药率在2．5％～10．7％。结论泌尿系统感染主要病原菌为肠杆菌和肠球菌,由于各病原菌均存在不同程度的耐药性,临床医师应根据尿液细菌培养结果,科学合理使用抗菌药物。