HP17, a new pigment-like antibiotic produced by a new strain of Spirillospora

Spirillospora strain 719 produces several antibiotics. On solid and liquid media, a deep red pigment is formed and diffuses throughout the culture. It was extracted with methanol from the mycelium cake and from the fermentation broth after precipitation at pH 2 and purified using TLC and HPLC. Its u.v. absorption spectrum and its physicochemical characteristics place this antibiotic in the 3.3.2.2.8 of the Berdy et al. classification. In most respects, it resembles proteinaceous pigment from Spirillospora 1655 and 1309-b that was studied and named spirillomycin. However, HP17 differs from spirillomycin principally in molecular weight and chemical nature.     

AH7, a non-polyenic antifungal antibiotic produced by a new strain of Streptosporangium roseum

An antibiotic (AH7) produced by Streptosporangium roseum strain 214 was investigated. This compound was extracted with chloroform from the filtrate culture and purified using thin-layer chromatography and high pressure liquid chromatography procedures. The antibiotic strongly inhibited the growth of several strains of fungi and bacteria known to be plant and human pathogens. This compound differed from all other antibiotics known to be synthesized by Streptosporangium spp. Some of its chemical and physical properties resembled those of maytansines produced by Nocardia but the antibiotic AH7 has only antibacterial and antitumoral activities.     

Genetic reconstruction of protozoan rRNA decoding sites provides a rationale for paromomycin activity against Leishmania and Trypanosoma

Aminoglycoside antibiotics target the ribosomal decoding A-site and are active against a broad spectrum of bacteria. These compounds bind to a highly conserved stem-loop-stem structure in helix 44 of bacterial 16S rRNA. One particular aminoglycoside, paromomycin, also shows potent antiprotozoal activity and is used for the treatment of parasitic infections, e.g. by Leishmania spp. The precise drug target is, however, unclear; in particular whether aminoglycoside antibiotics target the cytosolic and/or the mitochondrial protozoan ribosome. To establish an experimental model for the study of protozoan decoding-site function, we constructed bacterial chimeric ribosomes where the central part of bacterial 16S rRNA helix 44 has been replaced by the corresponding Leishmania and Trypanosoma rRNA sequences. Relating the results from in-vitro ribosomal assays to that of in-vivo aminoglycoside activity against Trypanosoma brucei, as assessed in cell cultures and in a mouse model of infection, we conclude that aminoglycosides affect cytosolic translation while the mitochondrial ribosome of trypanosomes is not a target for aminoglycoside antibiotics.     

HM17, a new polyene antifungal antibiotic produced by a new strain of Spirillospora

H. HACÈNE, K. KEBIR, D. SID OTHMANE AND G. LEFEBVRE. 1994. An antifungal antibiotic (HM17) was obtained from a new isolate classified to the genus Spirillospora on the basis of its chemical and morphological properties. On solid media this antibiotic strongly inhibited the growth of strians of Fusarium oxysporum formae speciales albedinis, Botrytis cinerea, Gaeumaniomyces graminis and several other fungi known to be plant and human pathogens. Antifungal activity in culture collection strains of Spirillospora has not so far been reported. The u.v. absorption spectrum and physico-chemical characteristics place HM17 in the methylpentaene sub-group of polyene macrolides. HM17 is different from other known methylpentaenes. This is the first report of polyene production by a Spirillospora.     

Protoplast fusion in Streptomyces fradiae strains producing neomycin and tylosin]

Interspecies fusion of protoplasts of the Streptomyces fradiae strains producing neomycin (an aminoglycoside antibiotic) and tylosin (a macrolide antibiotic) was performed with a view to isolate strains producing novel antibiotics. Fusion of the protoplasts of the neomycin- and tylosin-producing strains labelled by the resistance to monomycin and lincomycin, respectively, caused no formation of stable strains producing antibiotics differing in chromatographic mobility from the antibiotics produced by the initial strains. In fusion of the protoplasts of the unlabelled strains, heat-inactivated protoplasts of the active line of one strain (donor) and native protoplasts of the inactive line of the other strain (recipient) were used. When the neomycin-producing culture was used as a recipient the fusion led to formation of strain 195-34 producing antibiotics of the benzo(a)anthraquinone group. One of these antibiotics, i.e. antibiotic 34-I, proved to be a novel biologically active substance. After regeneration of the protoplasts of the initial strains, no stable strains producing antibiotics differing from neomycin and tylosin were isolated.     

Analysis of the etiological structure and dynamics of antibiotic resistance in the causative agents of suppurative inflammatory diseases in the patients at a large general hospital

The structure of the causative agents isolated from patients with pyoinflammatory infections in 1980-1983 was analysed. It was shown that the surgical and urological infections were mainly caused by gram-negative bacteria. The other pyoinflammatory infections were mainly due to gram-positive cocci. A relatively high frequency of the strains of gram-negative bacteria, especially among Pseudomonas spp. and Enterobacter spp., resistant to aminoglycoside antibiotics, such as gentamicin, sisomycin and tobramycin with preserved sensitivity to amikacin and netilmicin in the majority of the strains was shown. Among the beta-lactam antibiotics cephotaxim and cephalotin were most active against gram-negative bacteria and staphylococci, respectively. The majority of the antibiotic resistant strains of gram-negative bacteria had analogous structures and levels of resistance to 7-12 antibiotics which might indicate the occurrence of 1-2 resistance plasmids among the clinical strains.     

Mechanism of antibiotic resistance in Mycobacterium intracellulare

The mechanism of resistance of Mycobacterium intracellulare strain 103 and other clinical isolates to a variety of drugs including aminoglycoside and peptide antibiotics was investigated. Enzymatic inactivation of aminoglycoside and peptide antibiotics could not be demonstrated. Ribosomes of the strain were found to be sensitive to the antibiotics. The levels of resistance of strain 103 and other clinical isolates decreased dramatically when the culture medium was changed from Dubos agar to Tween 80-containing agar. These results suggest that a permeability barrier is the reason for naturally occurring resistance in M. intracellulare.     

Biosurfactant production by a new Pseudomonas putida strain

Observation of both tensio-active and emulsifying activities indicated that biosurfactants were produced by the newly isolated and promising strain Pseudomonas putida 21BN. The biosurfactants were identified as rhamnolipids, the amphiphilic surface-active glycolipids usually secreted by Pseudomonas spp. Their production was observed when the strain was grown on soluble substrates, such as glucose or on poorly soluble substrates, such as hexadecane, reaching values of 1.2 g l(-1). When grown on hexadecane as the sole carbon source the biosurfactant lowered the surface tension of the medium to 29 mN m(-1) and formed stable and compact emulsions with emulsifying activity of 69%.     

On the interaction between heparin and some aminoglycoside antibiotics

An interaction between the aminoglycoside antibiotics and heparin wherein charge transfer complexes are formed has been investigated to determine the degree of inhibition of antibacterial function of the antibiotic in the complexed form.Minimum inhibitory concentration (MIC) values have been obtained for the action of the aminoclycoside antibiotics tobramycin, gentamicin, amikacin, kanamycin, and streptomycin, on a sensitive strain ofE. coli. Growth curves ofE. coli determined at concentrations of these antibiotics just below the MIC demonstrated significant lengthening of the lag phase relative to control growth curves generated in the absence of antibiotic. Heparin (1 U ml^–1 and 10 U ml^–1) had no effect on control growth curves; however, particularly at the higher concentration, it reduced the effect on the lag phase produced by the aminoglycoside antibiotics. Thus kanamycin, gentamicin, and tobramycin were most affected, while amikacin and streptomycin were least affected. The rank order of inhibition of antibiotic activity by interaction with heparin was in qualitative agreement with previously published figures for the degree of complexation between antibiotics and heparin.     

Aminoglycoside-3'-phosphotransferase I from aminoglycoside-polyresistant strain E. coli 182]

An aminoglycoside-3'-phosphotransferase I catalyzing phosphorylation of some aminoglycoside antibiotics with the 3'-hydroxyl group has been purified from the cells of aminoglycoside resistant strain E. coli 182 by competitive affinity chromatography on neomycin-Sepharose and gel-filtration on Sephadex G-100. The product of enzymatic phosphorylation of kanamycin A was isolated and identified as kanamycin-3'-phosphate by NMR, thin-layer chromatography and chemical characterization. The kinetic properties of the enzyme were studied. The pH-optimum was between 7,8--8,0; the [S]0.5 values for kanamycin, neomycin and paromomycin were 2.10(-5) M, the energy of activation was 15,9 kcal/mol. The bivalent cations were required for activity of the enzyme, Mg2+ was the most effecient. The relative aminoglycoside antibiotics containing no 3'-hydroxyl group were competitive inhibitors of the enzyme activity with Ki values close to [S]0.5.     

Molecular characterization of InJR06, a class 1 integron located in a conjugative plasmid of Salmonella enterica ser. Typhimurium.

The presence of class 1, 2, and 3 integrons was investigated in four pediatric isolates of Salmonella enterica ser. Typhimurium (S. Typhimurium). A class 1 integron was detected in one S. Typhimurium strain, the only one that also showed resistance to various aminoglycoside antibiotics. This integron, called InJR06, and the aminoglycoside resistance determinants were located in pS06, a large (> or = 55 kb) conjugative plasmid. A single mobile cassette (encoding the aminoglycoside adenylyltransferase ANT(3')-Ia) was detected in the variable region of InJR06, while the architecture of the attI1 and attC sites was conserved.     

Effect of aminoglycoside antibiotics on the autolytic enzyme of Streptomyces griseus

The isolated cell wall of Streptomyces griseus 52–1 strain labelled with fluorescein isothiocyanate (FITC) and containing wall-bound autolytic enzyme was lysed as a function of different cations. The autolysis was accelerated by aminoglycoside antibiotics (streptomycin and the structurally closely related neomycin) which have a polycationic character. Since this strain is a streptomycin producer it is suggested that streptomycin may have a regulatory function on autolysis.     

Isolation of streptothricin resistant mutants from E coli K12, strain A19

Mutants with various levels of resistance to streptothricin were isolated from Escherichia coli K12, strain A19 after mutagenesis with N-methyl-N-nitro-N-nitroso-guanidine and ethylmethane-sulfonate. Nourseothricin, a mixture of streptothricin F and D was the selection agent. Spontaneous resistant mutants could not be found. The streptothricin-resistant mutant E. coli A19 Stcr 2/2/1 shows cross-resistance to some of the aminoglycoside antibiotics investigated, but no cross-resistance to chloramphenicol and chlortetracyclin. These results indicate similar mechanisms of action of streptothricin and aminoglycoside antibiotics.     

Comparative study of Streptomyces--producer of aminoglycoside antibiotics, monomycin and kanamycin, and the strain 344 obtained by fusion of their protoplasts by the method of restrictive total DNA fingerprinting]

The method of total DNA restriction finger prints was applied to the study of Streptomyces monomycini INA 1465 producing monomycin, Streptomyces kanamyceticus INA K-13 producing kanamycin and strain 344 isolated after fusion of the protoplasts of strain 1465 and K-13, which produced albofungin and chloralbofungin, aminoglycoside antibiotics. For preparing the finger prints of the strains splitting by endonucleases BamHI, PstI, PvuII, and BgIII was used. The finger prints showed that strain 344 was related to the strain of S. monomycini and markedly differed from the strain of S. kanamyceticus. Strain 344 was likely to result from reconstruction (probably 20-kb deletion) of the genome of S. monomycini INA 1465 induced by the preparation and regeneration of its protoplasts. The reconstruction could affect the genome area with localization of the genes involved in monomycin biosynthesis and monomycin resistance genes.     

A novel large plasmid carrying multiple beta-lactam resistance genes isolated from a Klebsiella pneumoniae strain

Klebsiella pneumoniae clinical isolates were selected according to the results of antibiotic susceptibility tests. Most of them were resistant to multiple antibiotics, including ampicillin, ceftazidime, cefotaxime and aminoglycosides. Large plasmids were observed in these Kl. pneumoniae strains by pulsed-field gel electrophoresis with S1 nuclease digestion. The Kl. pneumoniae strains investigated produced one to two extrachromosomal bands with a mobility corresponding to 97 approximately 145 kbp linear DNA molecules. A 100 kbp plasmid, designated pK1, was observed in the multiply resistant strain K250. pK1 had sequences homologous to both the TEM-1 and the aphD probe which were associated with beta-lactam and aminoglycoside resistance. pK1 was transformed into Escherichia coli strain DH5alpha and was found to confer resistance to ampicillin, ceftazidime, cefotaxime and kanamycin. A 8 kbp BamHI DNA fragment of pK1 that carried the ampicillin resistance gene (minimum inhibitory concentration > 1000 microgram ml-1) was cloned into the BamHI site of pACYC184. Sequence determination showed that this cloned fragment carried a TEM-1 gene. These findings suggest that pK1 is novel in that it appears to carry genes for resistance to ampicillin, cefotaxime and ceftazidime, as well as kanamycin.     

Characterization of a 30S ribosomal subunit assembly intermediate found in <Emphasis Type="Italic">Escherichia coli</Emphasis> cells growing with neomycin or paromomycin

Neomycin and paromomycin are aminoglycoside antibiotics that specifically stimulate the misreading of mRNA by binding to the decoding site of 16S rRNA in the 30S ribosomal subunit. Recent work has shown that both antibiotics also inhibit 30S subunit assembly in Escherichia coli and Staphylococcus aureus cells. This work describes the characteristics of an assembly intermediate produced in E. coli cells grown with neomycin or paromomycin. Antibiotic treatment stimulated the accumulation of a 30S assembly precursor with a sedimentation coefficient of 21S. The particle was able to bind radio-labeled antibiotics in vivo and in vitro. Hybridization experiments showed that the 21S precursor particle contained unprocessed 16S rRNA with both 5′ and 3′ extensions. Ten 30S ribosomal proteins were found in the precursor after inhibition by each drug. In addition, cell free reconstitution assays generated a 21S particle after incubation with either aminoglycoside. This work helps to define the features of the ribosome structure as a target for antimicrobial agents and may provide information needed for the design of more effective antibiotics.     

Detection of broad-spectrum aminoglycoside antibiotics through fluorescence-labeling aminoglycoside acetyltransferase(6')-Ii

Aminoglycosides are among the most commonly used antibiotics. The intensive use of aminoglycoside antibiotics has led to the problem of food contamination and the development of antibiotic-resistant bacteria. In the present study, we developed an effective method for easy sensitive detection of broad-spectrum aminoglycoside antibiotics. Aminoglycoside 6′-N-acetyltransferase family catalyzes the transfer of an acetyl group from acetyl coenzyme A (acetyl-CoA) to the 6′ amino group of the aminoglycoside, which is one of the most widespread determinants of aminoglycoside resistance. Because acetyl-CoA is naturally present only in living organisms, it is expected that the enzyme can bind with aminoglycoside antibiotics without catalysis in vitro. The enzyme was mutated for the introduction of a cysteine residue to flexible loops close to the binding site, which was then labeled with thio-labeling reagent fluorescein-5-maleimide. The labeled enzymes were characterized with kinetic and binding studies of various known aminoglycoside antibiotics. The binding of the labeled enzyme with aminoglycoside antibiotics causes a conformational change of the enzyme, which subsequently changes the hydrophobicity and hydrophilicity environment of fluorescent labeling reagent resulting in emission of fluorescence. This study provides a sensitive detection method for residual aminoglycoside antibiotics and strategies to screen and discover new effective aminoglycoside antibiotics.     

Exploration of genes that encode a carbocycle-forming enzyme involved in biosynthesis of aminoglycoside antibiotics from the environmental DNA

2-Deoxy-scyllo-inosose (DOI) synthase is the enzyme participating in biosynthesis of 2-deoxystreptamine (DOS)-containing aminoglycoside antibiotics. The gene which encodes the enzyme can be a marker for screening of DOS-containing aminoglycoside-producer and exploration of its biosynthetic gene. Further, this enzyme is expected to be of use in industry, because it converts sugar into 6-membered carbocycle. In the present study, we identified 21 clones encoding DOI synthase from environmental DNA by degenerate PCR. They were clearly divided into two groups. One appeared to derive from actinomycetes, and the other from non-actinomycetes. The latter group was larger (17 clones) than the former (four clones) despite the fact that only one strain of non-actinomycete was identified for DOS-containing aminoglycoside production. This result indicates that there are still many unidentified non-actinomycetes for DOS-containing aminoglycoside biosynthesis. We showed the possibility of identification of novel aminoglycoside-producing non-actinomycete from soil, and for development of more efficient enzymes for industrial use.     

Regulated readthrough: a new method for the alternative tagging and targeting of recombinant proteins

We report here a new method for the alternative peptide tagging of recombinant proteins from mammalian cell lines. This method, which we called regulated readthrough, exploits the property of aminoglycoside antibiotics to promote translational readthrough of nonsense codons. The basic expression cassette includes a translational fusion between a gene of interest and a membrane targeting peptide, which are separated by a nonsense codon. In the presence of an aminoglycoside antibiotic, translational readthrough is promoted and results in the targeting of the fusion protein to the cell membrane, thus allowing the efficient flow cytometry-based isolation of cells expressing very high levels of recombinant protein. For downstream applications requiring the production of soluble recombinant protein, the cells are cultured in the absence of aminoglycoside, leading to an efficient translational termination. By combining different translation termination signals that exhibit various susceptibilities to aminoglycoside-mediated translational readthrough with flow cytometry capabilities, it is possible to use this technology for other applications such as functional library screening or monitoring the stability of recombinant protein production.