Cross-resistance in Cell Lines of Nicotiana sylvestris Selected for Resistance to Individual Antibiotics

Cell lines initially selected for resistance to the antibioticskanamycin, streptomycin and chloramphenicol, were each testedfor resistance to several different antibiotics. Only the kanamycinresistant lines showed any cross-resistance to other antibiotics.The three lines tested were resistant to streptomycin and neomycin,while one of them, KR103, was also resistant to chloramphenicolearly in its history, although this resistance was subsequentlylost. None of the lines showed any resistance to cycloheximide. Nicotiana sylvestris, cell culture, cross-resistance, antibiotics, chloramphenicol, kanamycin, streptomycin, neomycin, cycloheximide, cytoplasmic mutants, callus     

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.     

Control of Pleuropneumonia-like Organisms in Cell Culture

Mammalian cell culture systems were maintained free of mycoplasmas by using a 3-day agar plate test as a weekly routine to monitor the conditions of the cells. If contaminated cell cultures were found, they were discarded and replaced from a pleuropneumonia-like organism (PPLO)-free cell bank. PPLO-free lines were established by treatment with various antibiotics. The KB cell line was freed of mycoplasmas by treatment for 1 week with a mixture of chlortetracycline, kanamycin, and chloramphenicol. L-929 cells were cleared of contamination with either spectinomycin or tylosin, and a synovial cell line was cleared with lincomycin or tylosin. Each cell line, after eradication of the contaminant, was stored in liquid nitrogen. A number of agents were tested to determine minimal inhibitory concentration against three known and three unidentified mycoplasmas. Chlortetracycline and tetracycline were found to be highly active against all strains, whereas tylosin, spectinomycin, and lincomycin, though less active, were equally useful because of their low toxicity against cells. Kanamycin was highly active against three strains, but inactive at high levels against the KB cell contaminants. A disc plate test was used to check isolated cell contaminants for sensitivity to various agents.     

Elimination of Mycoplasma from various cell cultures

Elimination ofMycoplasma orale-I from chronically infected cell lines was achieved either by treatment with a mixture of antibiotics in a hypotonic solution, or with 10 vol % of anti -M.orale rabbit serum in tissue culture medium. The latter treatment was preferable in most cases, as it was practically harmless to the cells. Inactivation of this antiserum had no effect on its potency. The antibiotic-hypotonic treatment was rather destructive, but to a different degree for the various cell cultures. Both methods were equally useful for the treatment of a monkey kidney cell line contaminated with a mycoplasma strain related toM.hyorhinis. The available anti -M.hyorhinis rabbit serum was toxic for the monkey cells when not inactivated. The potency of the antiserum was rather low and even lower after inactivation. However, prolonged treatment successfully eliminated the mycoplasma. Pre-incubation of the inactivated anti -M.hyorhinis serum with tissue culture medium to which 10% non-inactivated calf serum had been added, favoured the elimination of the mycoplasma.During the treatment of contaminated cell cultures with single antibiotics a strain related toM.hyorhinis became resistant to chlortetracyclin.M.orale- I was found to be resistant to various single antibiotics.We are grateful to Professor Dr. A. Ch. Ruys (University of Amsterdam) and Dr. R. H. Leach (Mycoplasma Reference Laboratory, London) for helpful discussions and for identifying some of our mycoplasma strains; Dr. Leach also for kindly supplying us with his G. D. L.-strain. We thank Dr. H. Cohen and Dr. A. C. Hekker for their criticism and Mr. N. L. M. van Zwetselaar for his accurate technical assistance.     

Ribosomal resistance of an istamycin producer, Streptomycestenjimariensis, to aminoglycoside antibiotics

$\text{Streptomyces}\text{tenjimariensis}$ SS-939 was resistant to its own aminoglycoside antibiotics, istamycins, as well as kanamycin A, neamine, ribostamycin and butirosin A, but was susceptible to neomycin B, lividomycin A and streptomycin. This resistance to these antibiotics was found to be due to ribosomes of the strain.     

PRODUCTION OF AXENIC CULTURES OF MICROMONAS PUSILLA (PRASINOPHYCEAE) USING ANTIBIOTIC 1

Bacteria-free cultures of the prasinophyte Micromonas pusilla (Butcher) Monton and Parke, UTEX LB 991, were produced by intially determining the effects of several antibiotics on the growth of this alga and then using a combination of these antibiotics to eliminate associated bacteria, Micrononas pusilla was resistant ot penicillin G, neomycin, gentamicin and streptomycin at bactericidal concentrations but sensitive to chloramphenicol and polymixin B. Passage of M. pusilla through the sequence of antibioties penicillin G → neomycin → gentamician → kanamycin resulted in an axenic culture of M, pusilla this method should be suitable for producing axenic culture of other strains of M. pusilla.     

Aminoglycosides resistance in clinical isolates of Staphylococcus aureus from a University Hospital in Bialystok, Poland

Staphylococcus aureus obtained from a University Hospital in Poland were characterized in relation to resistance to aminoglycoside antibiotics and the distribution of the genes encoding the most clinically relevant aminoglycoside modifying enzymes (AMEs). Of a total of 118 S. aureus, 45 (38.1%) isolates were found to be resistant to at least one of the tested antibiotics. All aminoglycoside resistant isolates except one 44 (97.8%) were resistant to kanamycin. The majority of strains 37 (82.2%) and 32 (71.1%) expressed resistance to neomycin and tobramycin, respectively. Eleven strains (24.4%) were resistant to gentamicin or amikacin. All S. aureus strains were sensitive to netilmicin. The most prevalent resistance gene was aac(6')-Ie+aph(2') found in 13 (28.9%) strains and 12 (26.7%) isolates carried ant(4')-Ia gene, whilst aph(3')-IIIa gene was detected in only 7 (15.6%) isolates. Additionally, the ant(6)-Ia and str genes were detected in 14 (31.1%) and 2 (4.4%) strains, respectively. Ten (22.2%) strains resistant to amikacin, tobramycin, kanamycin or neomycin did not harbor any of the above-noted genes.     

Coresistance to Neomycin and Kanamycin by Mutations in an Escherichia coli Locus that Affects Ribosomes

Mutant strains resistant to neomycin or to kanamycin sulfate were isolated from Escherichia coli K-12. Nine mutants were analyzed; all were resistant to both antibiotics (about 150 and 100 mug/ml, respectively), and were designated nek. In the mutant strains, the ribosomes are changed from those of the parental strain; for when they were used in assays for polypeptide formation directed by polyadenylic acid or polycytidylic acid, coding fidelity in presence of the drugs was increased and inhibition of synthesis by the drugs was lessened. Mating experiments and transduction tests showed that all of the nine nek mutants are either closely linked or allelic, and the nek locus is closely linked to two genes-str (streptomycin) and spc (spectinomycin)-known to affect the 30S ribosome. The two nek mutants tested were recessive to the sensitive, wild-type allele. When the nek mutants were compared to the parental strain, pleiotropic effects of the nek mutations were observed. Resistance to low levels of streptomycin and spectinomycin was increased, whereas resistance to chloramphenicol was decreased. Also, the mutants were less able to adapt to high concentrations of lincomycin, and could no longer show phenotypic suppression of an arginine requirement by neomycin or kanamycin. Such pleiotropic effects are suggested to be the rule for mutations in genes that participate in the biosynthesis of a cellular organelle.     

6种微藻对氯霉素和硫酸新霉素敏感性研究

目的:探讨3种真眼点藻(点状魏氏藻(Visderia punctata)、波氏真眼点藻(Eustigmatos polyphem)、魏氏真眼点藻(Eustigmatos vischeri))和3种绿藻(栅藻(Scnedesmus sp.)、斜生栅藻(Scenedesmus obliqulis)、爪哇栅藻(Scenedesmus jaoaensis))对2种抗生素的敏感性.方法:采用藻液细胞计数法和藻细胞固体平板培养法研究了氯霉素和硫酸新霉素对6种微藻生长的影响.结果:液体培养,3种绿藻对氯霉素敏感性均高于硫酸新霉素,10μg·mL-1氯霉素即可明显抑制3种绿藻的生长(P＜0.05),而硫酸新霉素在浓度为200 μg·mL-1时才显示出明显抑制作用:3种真眼点藻对2种抗生素都不敏感.固体培养,除波氏真眼点藻外,其它5种微藻对氯霉素的致死浓度均为50μg·mL-1;波氏真眼点藻、栅藻、斜生栅藻和爪哇栅藻对硫酸新霉素的致死浓度分别为100 μg·mL-1、200μg· mL-1、50μg·mL-1和50μg·mL-1.结论:氯霉素可作为选育6种微藻抗性突变株的筛选剂.     

Occurrence of resistance to neomycin and kanamycin in Bacillus popilliae and certain serotypes of Bacillus thuringiensis: Mutation potential in sensitive strains

Serotypes of Bacillus thuringiensis and the commercial strain of Bacillus popilliae were examined for their inherent resistance to antibiotics and their mutation potential in respect to neomycin and kanamycin, the presence of which would preclude the use of plasmids marked by genes for resistance to the antibiotics. Clones on initial plates were detected by the recurrence of resistance colonies at superimposable sites on serial replicaplates containing the antibiotic. Susceptible strains were selected for the determination of their antibiotic-resistance mutation potential. Three varieties of B. thuringiensis were found to be doubly resistant, seven varieties were singly resistant (Neo^r), and three other varieties, including B. popilliae, were susceptible to both antibiotics. Estimates of mutation ratios revealed that three serotypes developed no resistant mutants to either antibiotics in populations as high as 3.0 × 10¹⁰; seven other serotypes developed no resistance to kanamycin in populations as high as 4.6 × 10⁹ cells. Three other serotypes exhibited mutation ratios as high as 1.6 × 10^?2. We were unable to determine the mutation ratio for B. popilliae.     

Transfer of a genetic marker from a megaplasmid of Anabaena sp. strain PCC 7120 to a megaplasmid of a different Anabaena strain.

The 410-kb alpha megaplasmid of the heterocyst-forming cyanobacterium Anabaena sp. strain PCC 7120 was found to bear the nucA gene that encodes a sugar-nonspecific nuclease. That gene was mutated by insertion of a cassette that confers resistance to neomycin. The resulting strain, AMP2, was mated with a streptomycin-resistant derivative of Anabaena sp. strain PCC 7118, a strain that does not form heterocysts. Cells resistant to both neomycin and streptomycin that were derived from such matings were found to bear the neomycin resistance cassette of the donor strain in a larger megaplasmid characteristic of the recipient strain and did not form heterocysts. This is the first example of transfer of a genetic marker directly between strains of cyanobacteria in which incontrovertible physical evidence of transfer has been obtained. DNA sequences homologous to the nucA gene were present in 13 heterocyst-forming cyanobacteria that were tested but in none of six diverse unicellular strains that were examined.     

Comparison of Chlamydia trachomatis serovar L2 growth in polarized genital epithelial cells grown in three-dimensional culture with non-polarized cells

A common model for studying Chlamydia trachomatis and growing chlamydial stocks uses Lymphogranuloma venereum serovar L2 and non-polarized HeLa cells. However, recent publications indicate that the growth rate and progeny yields can vary considerably for a particular strain depending on the cell line/type used, and seem to be partially related to cell tropism. In the present study, the growth of invasive serovar L2 was compared in endometrial HEC-1B and endocervical HeLa cells polarized on collagen-coated microcarrier beads, as well as in HeLa cells grown in tissue culture flasks. Microscopy analysis revealed no difference in chlamydial attachment/entry patterns or in inclusion development throughout the developmental cycle between cell lines. Very comparable growth curves in both cell lines were also found using real-time PCR analysis, with increases in chlamydial DNA content of 400-500-fold between 2 and 36 h post-inoculation. Similar progeny yields with comparable infectivity were recovered from HEC-1B and HeLa cell bead cultures, and no difference in chlamydial growth was found in polarized vs. non-polarized HeLa cells. In conclusion, unlike other C. trachomatis strains such as urogenital serovar E, invasive serovar L2 grows equally well in physiologically different endometrial and endocervical environments, regardless of the host cell polarization state.     

New Combination of Antibiotics for Treatment of Contaminated Specimens for Viral Studies

A study of a new combination of antibiotics (sodium oxacillin, colistin sulfate, and amphotericin B) has shown that 30 μg/ml of each in tissue culture maintenance medium can inhibit a group of the commonest bacterial and fungal contaminants while causing no observable cytopathology in rhesus monkey kidney, HeLa, or human amnion tissue cells. In addition, there was no inhibition of the titers of poliovirus, echovirus, coxsackievirus, and adenovirus in the three cell lines. Ten duplicate fecal samples, which remained contaminated with fungal or Pseudomonas organisms after treatment with penicillin and streptomycin, were treated with the new antibiotic combination. Contaminants were controlled in each, and a variety of viruses was recovered.     

Relationship between Spontaneous Aminoglycoside Resistance in Escherichia coli and a Decrease in Oligopeptide Binding Protein

Changes in the amount of oligopeptide binding protein (OppA) in spontaneous kanamycin-resistant mutants of Escherichia coli were investigated. Among 20 colonies obtained from 10⁸ cells cultured in the presence of 20 μg of kanamycin/ml, 1 colony had no detectable OppA and 7 colonies were mutants with reduced amounts of OppA. Sensitivity of wild-type cells to kanamycin increased slightly by transformation of the oppA gene, but the sensitivity of the mutants increased greatly by the transformation. A mutant with no OppA was found to be a nonsense mutant of the oppA gene at amino acid position 166. In a mutant having a reduced level of OppA, the reduction was due to the decrease in OppA synthesis at the translational level. These mutants were also resistant to other aminoglycoside antibiotics, including streptomycin, neomycin, and isepamicin. Isepamicin uptake activities decreased greatly in these two kinds of mutants. The results support the proposition that aminoglycoside antibiotics are transported into cells by the oligopeptide transport system, and that transport is an important factor for spontaneous resistance to aminoglycoside antibiotics.     

Studies on the mechanism of resistance of Pseudomonas aeruginosa to neomycin. II. Correlation between neomycin resistance and hemoprotein concentration

The cells of a newly isolated neomycin sensitive and neomycin resistant strains of Pseudomonas aeruginosa contained similar level of catalase activity. The difference spectrum of the cells revealed the presence of cytochrome c-554 and cytochrome b-560-. The presence of both cytochromes in the same molecular ratio in the cells and subcellular fractions indicates that they are tightly bound to the cytoplasmic membrane. The neomycin resistant strain contained five times less cytochromes than the sensitive strain. The lower cytochrome level in the neomycin resistant cells was found to be independent of the presence or absence of neomycin in the medium. The difference spectra of the cells and particulate fractions did not reveal any cytochromes of alpha-type.     

Isolation and characterization of a reservosome fraction from Trypanosoma cruzi

Resistance to different antibiotics was found in 26 of the 30 strains analyzed, more than 70% of the strains analyzed were resistant to carbenicillin and ampicillin and a significant correlation was found between the resistance to both antibiotics. Plasmids were found in 80% of the strains analyzed, and 11 different plasmid profiles were observed. The most common profile obtained had only a 21.2-kbp plasmid, a significant correlation was found between the presence of this plasmid and resistance to carbenicillin, although some exceptions could be detected. Plasmids were cured from a cephalothin resistant strain and reintroduced into the plasmid-free cell and into Escherichia coli DH5alpha, both strains gained resistance to this antibiotic.     

Cell wall components in Staphylococcus aureus with double resistance to gramicidin S and actinomycin D]

Cell walls of Staphylococcus aureus R9/80 resistant to gramicidin S and actinomycin D were investigated. The strain was isolated after passages of a previously isolated strain of S. aureus with resistance to gramicidin and definite changes in the cell walls, a medium with increasing concentrations of actinomycin being used for the passages. The data on the study of the cell walls of the strain with the double resistance were compared with the results of the investigation of the cell walls of the strain susceptible to gramicidin, the gramicidin resistant strain (initial for strain R9/80) and the actinomycin adapted strain that also showed changes in the cell walls. The cell walls of the resistant strains had no significant changes in the peptidoglycane and glucosamine levels, as well as in the peptidoglycane amino acid composition. Teichoic acids of all the strains had different levels of substitution of ribite by D-alanine (a factor influencing the negative charge of teichoic acids and the wall at large). It was noted that all the strains resistant to the tested antibiotics had lower levels of teichoic acids in the cell walls. The resistant cells showed some increase of the lipid component in the walls: from 1.6% in the susceptible strain to 2.1-2.9% in the resistant cells. The main trend of the changes in the resistance development was revealed to be the thickening of the cell wall and its consolidation. The development of resistance to gramicidin, actinomycin and to both the antibiotics provoked respectively a 2.4-, 4- and 5.4-fold increase of the content of the main cell component. i.e. peptidoglycane in the cell biomass. The barrier role of the cell walls in the resistant strains and their ability to bind the antibiotic is discussed.     

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.     

Role of Lipopolysaccharides in Antibiotic Resistance and Bacteriophage Adsorption of Escherichia coli K-12

Novobiocin-supersensitive (NS) mutants which could not grow on plates containing 40 mug or less of novobiocin per ml were isolated from Escherichia coli strain JE1011 (derived from E. coli K-12). Most of these NS mutants were found to have incomplete lipopolysaccharides (LPS), and they lack phosphate diester bridges in their backbone structure, with or without total loss of heptose, to which the phosphate diester is linked, and consequently lack external outer-core oligosaccharides. The phosphate diester bridges in the LPS backbone are apparently very important in forming a cell surface structure resistant to the penetration of antibiotics such as novobiocin, spiramycin, and actinomycin D. NS mutants, with incomplete LPS, lacking phosphates in their backbone structure were found to be resistant to phage T4, and those which also lacked heptose were resistant to phages T4 and T7. In contrast to the generally accepted idea that resistances to phages T3, T4, and T7 are linked genetically, no NS mutant was found to be resistant to T3. The possible structures of the receptors for T4 and T7 are discussed. The positions of novobiocin-supersensitive genes on the chromosome of several of the NS mutants defective in LPS were mapped. The genes were designated lpcA (between ara and lac) and lpcB (between 55 min and 60 min). The latter seemed to be a group of several related genes.     

Polypeptide synthesis directed by DNA as a messenger in cell-free polypeptide synthesis by extreme thermophiles,Thermus thermophilus HB27 and Sulfolobus tokodaii strain 7

Polypeptide synthesis at high temperature directed by single strand DNA as a messenger was investigated using cell-free extracts of an extremely thermophilic bacterium, Thermus thermophilus strain HB27, and a hyperthermophilic, acidophilic archaeon, Sulfolobus tokodaii strain 7. Aminoglycoside antibiotics enhanced the reaction; neomycin stimulated it most effectively when the extract of the thermophilic bacterium was used, and paromomycin was the best among the antibiotics tested for the extract of the hyperthermophilic archaeon. A common correlation was found between the stimulation of DNA-directed polypeptide synthesis and the misreading rate in RNA-directed polypeptide synthesis. Spermine stimulated the reaction directed by DNA like in the case of poly(Phe) synthesis directed by poly (rU). The cell-free systems can be used for direct production of proteins from genes in high throughput studies on the structural genomics of thermophilus.