Distribution of Streptomycin Resistant Strains of Erwinia amylovora in Egypt during 1988

A survey of the occurrence of strains of Erwinia amylovora resistant to streptomycin in certain Egyptian pear orchards was earned out during April and May 1988. Twenty-two isolates out of 604 isolates collected from 11 orchards showed resistance to streptomycin. All the streptomycin resistant (Str^r) strains isolated in the present work were resistant to high levels of streptomycin with minimal inhibitory concentrations ranging from 1000 to 3000 μg/ml. The occurrence of Str^r strains in Egypt is still limited and the population of resistant strains was at relatively low level. However, such occurrence of E. amylovora with resistance to streptomycin is a potentially serious situation.     

Enhancing Soybean Rhizosphere Colonization by Rhizobium japonicum

A study was conducted to seek means to increase the colonization of the rhizosphere of soybeans (Glycine max L. Merrill) by Rhizobium japonicum. For this purpose, a strain of R. japonicum that was resistant to benomyl, streptomycin, and erythromycin was used. The numbers of R. japonicum rose quickly in the first 2 days after soybean seeds were planted in soil and then rapidly fell. The decline was slower if the seeds were coated with benomyl. This fungicide reduced the numbers of bacteria and protozoa in the rhizosphere, but the effect became less or disappeared as the plants grew. In sterile soil inoculated with R. japonicum and a mixture of microorganisms, the numbers of R. japonicum were usually lower if protozoa were present than if they were absent. Nodulation and plant yield were increased by the addition of benomyl to soybean seeds sown in sterile soil inoculated with R. japonicum and a mixture of microorganisms. The addition of streptomycin and erythromycin to soil stimulated the growth of R. japonicum but inhibited other bacteria in the presence or absence of soybeans. The data indicate that colonization can be increased by the use of antimicrobial agents and R. japonicum strains resistant to those inhibitors.     

Phenotypic suppression by streptomycin of amber mutants in the ribonucleic Acid bacteriophage coat protein cistron

After mutagenesis with nitrosoguanidine or ultraviolet light, 298 streptomycin high-resistant and 98 streptomycin high-dependent mutants were isolated from HfrC Su⁻. They were tested for their ability to phenotypically suppress five different amber ribonucleic acid (RNA) bacteriophage mutants in the presence of streptomycin. The phage mutants are all in the coat protein, which is 129 amino acids long; the uracil-adenine-guanine codons were at the following positions: sus3 and amB2, 6; amB11, 50; amB21, 54; sus11, 70. Only sus3 and amB2 could be phenotypically suppressed by streptomycin; this was clearly demonstrated in nine mutant strains, seven str-HR and two str-HD. The suppression was always dependent upon added streptomycin and was dose-dependent in all cases. None of the mutants showed measurable suppression in absence of the drug. Among revertants to streptomycin independence from streptomycin-dependent strains that could show phenotypic suppression, most of those that were still resistant to streptomycin (10 μg or more) retained the capacity to show phenotypic suppression; whereas among those revertants sensitive to 10 μg of streptomycin or less, none retained the capacity. Eight different amber polar mutants (strong and weak) in gene 34 of phage T4 were also tested for pleiotypic suppression by streptomycin in all the streptomycin-resistant and -dependent strains isolated. No suppression was found in any of the 396 strains tested.     

Resistance ofBradyrhizobium japonicum strains to selected antibiotics

The majority of the 50Bradyrhizobium japonicum strains tested were resistant to ampicillin, kanamycin, streptomycin and tetracycline in concentrations below 100 g/ml but resistant to chloramphenicol in concentrations equal to or above 100 g/ml. Two strains had high levels of resistance to ampicillin and to streptomycin and six strains were very sensitive to several antibiotics.

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Résumé La majorité de la cinquantaine de souches deBradyrhizobium japonicum s'est révélée résistante à l'ampicilline, à la kanamycine, à la streptomycine et à la tétracycline aux concentrations intérieures à 100g/ml mais au chloramphenicol aux concentrations égales ou supérieures à 100 g/ml. Deux souches présentalent un haut degré de résistance à l'ampicilline et à la streptomycine. Six souches étaient très sensibles à plusleurs antibiotiques.

     

Strategies for adaptation to antibiotics in wild type <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> and in the strains with small colony phenotype

Emergence of ciprofloxacin stress-induced mutants in the cultures of a collection strain Pseudomonas aeruginosa ATCC 27853 and of two strains with a small colony phenotype, which were isolated from a swimming pool biofilm, was studied. In biofilm cultures of the small colony phenotype strains, which were already resistant to hypochlorite, prolonged incubation (up to 16 days) with sublethal ciprofloxacin concentrations was shown to result in emergence of the cells, which are resistant to the antibiotic and form colonies on media with rifampicin (100 μg/mL) and streptomycin (50 μg/mL). Under the same conditions, the mechanisms of temporary adaptation are switched on in the cells of strain ATCC 27853, which enabled its shortterm survival at an average level in liquid media and provided for colony formation on solid medium with ciprofloxacin (0.2 μg/mL). Only 20% of these colonies remained viable when transferred to a higher antibiotic concentration (2 μg/mL).     

Acquisition of rifabutin resistance by a rifampicin resistant mutant of Mycobacterium tuberculosis involves an unusual spectrum of mutations and elevated frequency

Background

Mutations in a small region of the rpoB gene are responsible for most rifamycin resistance in Mycobacterium tuberculosis. In this study we have sequentially generated resistant strains to first rifampicin and then rifabutin. Portions of the rpoB gene were sequenced from 131 randomly selected mutants. Second round selection resulted in a changed frequency of specific mutations.

Methods

Mycobacterium tuberculosis (strain Mtb72) rifamycin resistant mutants were selected in vitro with either rifampicin or rifabutin. One mutant R190 (rpoB S522L) selected with rifampicin had a rifampicin MIC of 32 μg/ml but remained sensitive to rifabutin (MIC<0.8 μg/ml). This mutant was subjected to a second round of selection with rifabutin.

Results

All 105 first round resistant mutants derived from the parent strain (Mtb72) screened acquired mutations within the 81 bp rpoB hotspot. When the rifampicin resistant but rifabutin sensitive S522L mutant was subjected to a second round of selection, single additional rpoB mutations were identified in 24 (92%) of 26 second round mutants studied, but 14 (54%) of these strains contained mutations outside the 81 bp hotspot (codons 144, 146, 148, 505). Additionally, spontaneous rifabutin resistant mutants were produced at >10 times the frequency by the S522L mutant than the parent strain.

Conclusion

First round selection of mutation S522L with rifampicin increased the frequency and changed the spectrum of mutations identified after selection with rifabutin.     

Conditions for mutagenesis by N-methyl-N′-nitro-N-nitrosoguanidine and relationships of A. tumefaciens mutants to crown-gall tumor induction

Optimum mutagenesis of Agrobacterium tumefaciens by N-methyl-N′-nitro-N-nitrosoguanidine occurred at pH 6.5 using 250 μg/ml of the mutagen for 3 h at 30°. Antibiotic-resistant mutants and amino acid auxotrophs were selected and scored for crown-gall tumor-inducing ability on Helianthus annuus (sunflower). Mutants resistant to neomycin, kanamycin or rifampicin were not directly affected in their tumor-inducing ability. Mutants that were resistant to neomycin were also resistant to kanamycin and vice versa. Various amino acid auxotrophs varied in virulence. Some of the auxotrophs that required histidine, leucine or tryptophan had simultaneously lost their virulence. The alteration of virulence of the organism is not dependent on its growth since the avirulent auxotrophs when supplemented with the amino acid requirement grew in vivo almost as well as the prototrophic strains and yet remained avirulent.     

R-Plasmids in Pasteurella multocida.

Multiple drug-resistant strains of Pasteurella multocida were associated with a high incidence of fatal pneumonia in feedlot cattle. A representative strain, CAH160, resistant to tetracycline (Tc), streptomycin (Sm), and sulfonamide (Su) was studied. The minimal inhibitory concentration (MIC) of Tc was 32 μg/ml while Sm had an MIC of 256 μg/ml. Plasmid DNA was isolated from CAH160 by cesium chloride-ethidium bromide centrifugation. Agarose gel electrophoresis showed that at least three distinct species of plasmid DNA were present. DNA isolated from CAH160 was used to transform Escherichia coli K12 strain C600 r_k^?m_k^?. Transformants resistant to Tc; to Sm, Su; and to Tc, Sm, Su were obtained. Contour length measurements of plasmid DNA isolated from transformant cells showed that Tc resistance was associated with a 3-Mdal plasmid (pSR10), while Sm, Su resistance resided on a 2.7-Mdal molecule (pSR11). More than 20% of the transformants were resistant to Tc, Sm, Su and contained both plasmid species. In E. coli the MIC of Tc was 256 μg/ml and that of Sm was 64 μg/ml. The buoyant density of pSR10 was 1.699 g/cm³, while the density of pSR11 was 1.709 g/cm³.     

Benomyl Tolerance of Ten Fungi Antagonistic to Plant-parasitic Nematodes

Ten strains of fungi were tested for tolerance to the fungicide benomyl. Verticillium chlamydosporium strain 2 did not grow in the presence of benomyl; Drechraeria coniospora strains 1 and 2 and Chaetomium sp. tolerated only 0.1 μg benomyl/ml medium; Acremonium bacillisporum, an unidentified fungus, and Phoma chrysanthemicola uniformly grew at 1 μg/ml, but some hyphae grew at higher benomyl concentrations; Fusarium sp. tolerated 475 μg/ml, but some hyphae grew on medium amended with 1,000 μg/ml; Verticillium lecanii and V. chlamydosporium strain 1 routinely tolerated 1,000 μg/ml. Fungi generally grew more slowly at higher than at lower benomyl concentrations. Strains with elevated tolerance to benomyl were selected from Acremonium bacillisporum, Drechmeria coniospora, Fusarium sp., and an unidentified fungus. These strains retained the increased tolerance after repeated transfers on unamended medium.     

Isolation of mutants of Escherichia coli uncoupled in oxidative phosphorylation using hypersensitivity to streptomycin

Mutants of Escherichia coli, harbouring the uncA401 or uncB402 alleles, were found to take up streptomycin more rapidly than the coupled parent strains. The increased rate of uptake results in greater sensitivity of the uncoupled strains, compared to the parent strains, to low concentrations of streptomycin. Studies with unc⁺ revertants showed that hypersensitivity to streptomycin is attributable to the mutation causing uncoupling. The uptake of streptomycin in an unc^? strain is abolished by addition of the chemical uncoupler carbonylcyanide m-chlorophenylhydrazone. The phenotype of hypersensitivity to streptomycin can be used as a selection procedure for the isolation of uncoupled strains. In an experiment reported here, nine out of 12 strains isolated as being sensitive to streptomycin (at 2.5 μg/ml), were found to be unable to grow on succinate as a sole source of carbon. Five of the nine Suc^? strains were found to be uncoupled in oxidative phosphorylation, and two of the five uncoupled strains lacked Mg²⁺-ATPase activity. The mutations causing uncoupling were cotransducible with the ilv genes.     

Survival and multiplication ofRhizobium japonicum strains in silt loam

Summary Antibiotic resistant mutants 8-0 Str^R, 110 Tet^R and 138 Kan^R derived from wild typeRhizobium japonicum strains were inoculated into silt loam soil to cell concentrations greater than 2×10⁸/g of soil. Population changes were monitored using antibiotic media and strain identification was done using immunodiffusion assay on microcores of soil. Immunodiffusion bands formed by the mutant strains with homologous antisera essentially duplicated bands formed by the parent strain. Strains 110 Tet^R and 8-0 Str^R had cross reacting antigens whereas antigens of strain 138 Kan^R reacted only with the homologous antiserum. Populations ofR. japonicum strains introduced into sterile soil increased over a period of four weeks under both single and mixed culture inoculations. All populations decreased by the end of six weeks and thereafter remained constant. When theseR. japonicum strains were introduced into non-sterile soil, the population did not increase over the initial population added. Population decreased gradually for two weeks and then maintained thereafter. It was possible to recover very low populations of antibiotic resistantR. japonicum strains from both sterile and unsterile soils using media containing specific antibiotics. Detection ofR. japonicum strains by immunodiffusion was accomplished only when the population was 10⁹ cells/g of soil. The method using antibiotic resistant mutants permitted an evaluation of the interactions of variousR. japonicum strains in soil with respect to their survival and multiplication.     

Resistance to Azole Drugs in Neurospora crassa

Staben, C. 1995. Resistance to azole drugs in Neurospora crassa. Experimental Mycology 19: 163-165. Neurospora crassa was susceptible to azole drugs: ketoconazole (MIC 1 μg/ml), fluconazole (MIC 5 μg/ml), and SCH39304 (MIC 5 μg/ml). Mutants of N. crassa resistant to ketoconazole were selected and genetically characterized. The seven characterized resistance mutations represented at least four genetic loci. Some mutants, but not all, were also resistant to fluconazole and to SCH39304.     

The influence of serum components on the growth and mutation of Chinese hamster cells in medium containing 8-azaguanine

Low concentrations (?20 μg/ml) of 8-azaguanine are 1000 fold more toxic to V79 Chinese hamster cells in medium containing 10% dialyzed fetal calf serum than in medium containing 10% undialyzed serum. Serum enzyme activity that converts AG to nontoxic 8-azaxanthine degrades AG at the same rate, whether or not the serum is dialyzed. However, cytotoxicity results similar to those obtained with US were produced in medium containing DS and 2.5 μg of hypoxanthine (HX)/ml (DSH). Therefore, serum HX is considered to be responsible for the relatively low cytotoxicity of AG in medium containing US. Colonies that arose in medium containing AG were isolated and characterized. Those that remained resistant to AG (40 μg/ml) and sensitive to aminopterin in the presence of HX and thymidine (HAT) were considered mutants; nonmutants were sensitive to AG and resistant to HAT. Colonies isolated from medium containing DSH of US and low concentrations of AG were not mutants, but those from medium containing high concentrations (? μg/l) of AG were mutants. Spontaneous and N-methyl-N′-nitrosoguanidine induced mutants were detectable in medium containing DSH without replating the cells prior to adding AG (?30 μg/ml), but in order to detect MNNG induced mutations in medium containing DS replating was essential. In DS, the mutation frequency increased as an exponential function of the toxicity of MNNG, but remained two orders of magnitude lower than the induced mutation frequencies that occurred in DSH, HX, in DSH or US, produced profound effects, other than interference with AG toxicity, that distort the results of mutagenesis assays. To study mutation using AG resistance as the endpoint, it is essential to use dialyzed serum.     

In vitro cultivation of Hymenolepis diminuta: effect of antibiotics on the growth of three-day-old worms removed from the rat

The effects of several antibacterial or antifungal antibiotics on the growth of 3-day-old Hymenolepis diminuta cultivated in vitro were investigated. Worms were recovered from the rat and cultured in roller tubes for 4 days without a medium change. The medium contained horse serum, yeast extract, and liver extract; the gas phase was 5% CO₂ in N₂. It was found that penicillin and streptomycin did not inhibit the growth of worms at concentrations lower than 5000 units and μg/ml of medium, respectively. Cycloheximide was toxic to H. diminuta, retarding or inhibiting growth at levels higher than 1.5 μg/ml. The antifungal antibiotics nystatin and amphotericin B did not affect worm growth at concentrations lower than 1000 units and 123 μ/ml of medium, respectively.The use of penicillin and streptomycin, and nystatin or amphotericin B can be recommended for the control of bacterial and fungal contaminants in the cultivation of H. diminuta removed from the rat.     

In Vitro Effect of Rifampin on Mycobacteria

Rifampin inhibited 20 strains of Mycobacterium tuberculosis in concentrations of 0.005 to 0.02 mug/ml in 7H-9 broth with Tween 80 and killed all or nearly all of the inoculum in four to eight times greater concentrations. In the same medium without Tween 80, as well as on 7H-10 agar, about 16 to 64 times these amounts were required to produce the same effect. Rifampin was also active against M. kansasii and some of the nonchromogenic mycobacteria. The incidence of mycobacterial cells resistant to rifampin within the cultures studied was in the range of one to four per 10(8) to 10(9) colony-forming units with concentrations of 4 to 125 mug of rifampin per ml. Only one of the Battey cultures and that of M. fortuitum yielded cells resistant to rifampin at 125 mug/ml but not at 500 mug/ml. The same strains yielded more than double that number of organisms resistant to streptomycin and up to 100 times more organisms resistant to isoniazid. All three drugs stopped the growth or reduced the mycobacterial population in growing cultures after contact for 24 to 48 hr. Complete inhibition of growth was produced by rifampin at 1.0 mug/ml in an average of 6 days and by streptomycin at 5.0 mug/ml in 3 days. After an average contact of 10.7 days with rifampin, five of seven strains resumed growth and all strains began regrowth after exposure to streptomycin for 9.4 days. The marked susceptibility of M. tuberculosis and of atypical mycobacteria to rifampin in vitro and the relatively low incidence of resistant mutants suggests that this agent may have clinical usefulness in the treatment of tuberculosis and some other mycobacterioses.     

Mutations in Chlamydomonas reinhardtii Conferring Resistance to the Herbicide Sulfometuron Methyl

Chlamydomonas reinhardtii mutants resistant to the herbicide sulfometuron methyl (SM) were isolated and characterized. Growth of C. reinhardtii is sensitive to inhibition by SM at a concentration of 1 micromolar. Four mutants resistant to 10- to 100-fold higher concentrations were isolated. All possess a form of acetolactate synthase (ALS) whose specific activity in cell extracts is 100- to 1000-fold more resistant to SM than is the specific activity of wild-type enzyme. Only one mutant had abnormally low ALS specific activity in the absence of SM. All mutations were inherited as single lesions in the nuclear genome and were expressed in heterozygous diploids. The mutations in two strains mapped to linkage group IX about 30 centimorgans from streptomycin resistance and on the same side of the centromere, and in genetic crosses between mutants no segregation was observed. Accordingly, all mutations are tentatively assigned to gene smr-1. Herbicide resistance appears to be suitable as a selectable marker for molecular transformation in this organism.     

Unclassified Mycobacteria Isolated from Human Suspect Tuberculosis Cases in Newfoundland: Preliminary Studies on Fifteen Strains

Unclassified mycobacteria were isolated from 36 of 35,555 clinical specimens cultured for M. tuberculosis. The majority of isolations were from patients suspected of having tuberculosis and from whom repeat attempts at culture failed to yield typical tubercle bacilli. Fifteen strains thus far studied were not capable of causing generalized tuberculosis in guinea pigs, and all were highly resistant to the commonly employed antituberculous therapeutic agents. Eleven of the 15 strains were resistant to 100 or more μg./ml. of streptomycin; 12 strains were resistant to 25 or more μg./ml. of para - aminosalicylic acid; and all 15 showed growth in the presence of 50 or more μg./ml. of isoniazid. All strains were niacin-negative and catalase-positive. In the absence of other cultural findings, isolation of anonymous mycobacteria poses a major problem, especially in those cases in which the clinical and radiographic findings are typically those of tuberculosis.     

Resistance system of Mycobacterium bovis B.C.B. to aminoglycoside- and peptide-antibiotics. Comparison of resistant phenotypes between Mycobacterium tuberculosis and Mycobacterium bovis

The resistance system of Mycobacterium bovis (B.C.G.) to aminoglycoside-and peptide-antibiotics has been studied. The phenotype of mutants isolated from the parent B.C.G. strain by a single-step selection with an antibiotic were classified into the following three types: (1) resistant only to a low concentration (200 μg/ml) of kanamycin in Ogawa egg medium (k1R); (2) resistant to a low concentration (200 μg/ml) of viomycin and of capreomycin (2R); and (3) resistant to a high concentration (1,000 μg/ml or more) of kanamycin and low concentrations (100 to 200 μg/ml) of lividomycin and of paromomycin (KR). The mutants showing these phenotypes, k1R, 2R, and KR, were isolated from the parent strain by inoculating the strain into media containing 100 μg/ml of kanamycin, and 100 μ/g/ml of viomycin or capreomycin, and 1,000 μg/ml of kanamycin, respectively, at rates of 10^?5-10^?6, 10^?5-10^?6, and 10^?6-10^?7, respectively, in a total viable population of the parent strain. Unlike in the case of M. tuberculosis, no mutant could be isolated from the parent strain by use of enviomycin, lividomycin, and/or paromomycin. In contrast to the fact that quadruply resistant mutants were isolated directly from the parent H37Rv strain of M. tuberculosis, such mutants could be isolated only by two-step selections. Furthermore, the phenotypes of the quadruply resistant mutants were those showing a higher resistance or a broader spectrum than expected by the addition of phenotypes of individual mutations. In addition, it was shown that, in contrast to the fact that hextuply resistant mutants could be isolated directly from the parent strain of M. tuberculosis, such mutants were not isolated directly from the parent B.C.G. strain, but could be isolated only after pre-incubation of the strain on a medium containing Tween 80.     

Susceptibility of N. gonorrhoeae to Antibiotics: A Study of 200 Consecutive Strains Isolated in Winnipeg in 1962

Gonorrhea has increased in incidence over the past five years in Manitoba as elsewhere. Cases which did not respond to routine penicillin treatment were noted to be occuring more often. In 1948, strains of N. gonorrhoeae isolated in Canada were all sensitive to 0.06 unit of penicillin per c.c. in vitro. Commencing in May 1962, strains of N. gonorrhoeae were isolated from 100 patients of each sex attending the clinic at the St. Boniface General Hospital. Eighteen per cent required concentrations of more than 0.05 unit of penicillin per ml. to inhibit growth; 31% were not inhibited by the 2 μg. disc of dihydrostreptomycin; but only one strain was found resistant to sulfisoxazole and none to oxytetracycline. Results of penicillin treatment were markedly less successful in the patients with strains that demonstrated reduced penicillin sensitivity. Use of streptomycin and a sulfonamide proved to be a satisfactory substitute. Intramuscular oxytetracycline was less successful despite the laboratory findings.