Spontaneous Resistant Mutants Research Articles

Occurrence of quinolone resistance in Staphylococcus aureus from nosocomial infection.

Among 63 Staphylococcus aureus isolates (one isolate per one patient) counted from infections (from August to November 1991) in hospital T., eight exhibited resistance to fluoroquinolones. Seven of these quinolone-resistant isolates were multiply- and methicillin-resistant S. aureus (QR-MRSA). The results of phage-, plasmid- and genotyping (pulsed field electrophoresis) revealed that six different strain-clones of these MRSA were spread in the hospital. In vitro spontaneous mutants resistant to fluoroquinolones are 10-100-fold more frequent in MRSA than in other S. aureus when selected on isosensitest-agar containing 1 microgram/ml of ciprofloxacin. However, the same mutant frequencies were found in strain 8325-4 with and without the mecA-determinant. The resistance phenotype was stable over 30 generations of subculture in nutrient broth as well in natural quinolone resistant MRSA as in mutants of other types of S. aureus selected in vitro. The phenotypic association of quinolone resistance and MRSA is rather likely due to a higher frequency of spontaneous resistant mutants which are present in natural populations of MRSA. Data of chemotherapy prior to the isolation of S. aureus show that three of seven patients from whom QR-MRSA were isolated were treated with a quinolone. In eight cases of infections with non-MRSA and quinolone treatment the isolated S. aureus strains were in vitro sensitive to quinolones.

Yeast mitochondrial DNA mutators with deficient proofreading exonucleolytic activity.

The MIP1 gene which encodes yeast mitochondrial DNA polymerase possesses in its N-terminal region the three motifs (Exo1, Exo2 and Exo3) which characterize the 3'-5' exonucleolytic domain of many DNA polymerases. By site directed mutagenesis we have substituted alanine or glycine residues for conserved aspartate residues in each consensus sequence. Yeast mutants were therefore generated that are capable of replicating mitochondrial DNA (mtDNA) and exhibit a mutator phenotype, as estimated by the several hundred-fold increase in the frequency of spontaneous mitochondrial erythromycin resistant mutants. By overexpressing the mtDNA polymerase from the GAL1 promoter as a major 140 kDa polypeptide, we showed that the wild-type enzyme possesses a mismatch-specific 3'-5' exonuclease activity. This activity was decreased by approximately 500-fold in the mutant D347A; in contrast, the extent of DNA synthesis was only slightly decreased. The wild-type mtDNA polymerase efficiently catalyses elongation of singly-primed M13 DNA to the full-length product. However, the mutant preferentially accumulates low molecular weight products. These data were extended to the two other mutators D171G and D230A. Glycine substitution for the Cys344 residue which is present in the Exo3 site of several polymerases generates a mutant with a slightly higher mtDNA mutation rate and a slightly lower 3'-5' exonucleolytic activity. We conclude that proofreading is an important determinant of accuracy in the replication of yeast mtDNA.

Mechanisms and frequency of resistance to temafloxacin

The bactericidal activity of temafloxacin and other fluoroquinolones is attributed to inhibition of bacterial DNA gyrase (topoisomerase II), an enzyme that regulates supercoiling and uncoiling of DNA. Because of this unique bactericidal mechanism of action, resistance to fluoroquinolones is limited to spontaneous mutations. In vitro studies were conducted to determine the frequency at which spontaneous resistance to temafloxacin occurs and to determine whether cross-resistance to other antimicrobial agents develops. In 13 strains of bacteria, the frequency of spontaneous resistance mutation to concentrations of temafloxacin at four and eight times the minimal inhibitory concentration (MIC) ranged from <1 × 10 −10 to 1.4 × 10 −7. Temafloxacin demonstrated a lower frequency of resistance to both methicillin-resistant and methicillin-sensitive Staphylococcus aureus compared with ciprofloxacin and ofloxacin. Cross resistance with other fluoroquinolones was observed.

CysB and cysE mutants of Escherichia coli K12 show increased resistance to novobiocin

Mutations in the cysB and cysE genes of Escherichia coli K12 cause an increase in resistance to the gyrase inhibitor novobiocin but not to coumermycin, acriflavine and rifampicin. This unusual relationship was also observed among spontaneous novobiocin resistant (Novr) mutants: 10% of Novr mutants isolated on rich (LA) plates with novobiocin could not grow on minimal plates, and among those approximately half were cysB or cysE mutants. Further analyses demonstrated that cysB and cysE negative alleles neither interfere with transport of novobiocin nor affect DNA supercoiling.

Spontaneous frequency of 6‐thioguanine resistant mutants in CHO‐AS52 cells after prolonged culturing in the absence of selective agents

The Escherichia coli xanthine-guanine phosphoribosyl transferase (XPT) containing Chinese hamster ovary (CHO-AS52) cells were studied in our laboratory to evaluate its general applicability in genotoxicity testing of industrial chemicals. Our initial effort was to evaluate the stability of the 6-thioguanine sensitive phenotype. The CHO-AS52 cells were cloned and cultured continuously for over 600 days in the absence of any selective agents. The spontaneous 6-thioguanine resistant mutant frequency was quantified periodically. The frequency was found to increase continuously with time from approximately 20 x 10(-6) to a plateau of approximately 250 x 10(-6) in 122 days. The mutant frequency fluctuated around the plateau value up to the last day of our study, at 648 days after cloning. A similar observation was made for cells that were recloned or treated with aminopterin (to selectively kill 6-TG resistant mutants) at approximately 450 days after the initial cloning. Our data suggest that the xpt gene was stably incorporated in the CHO cells.

Effect of bacterial biomass on cadmium concentration in liquid medium and its toxicity towards bacteria

The efficiency of cadmium (Cd) removal from liquid medium by Pseudomonas sp. strain sensitive to this heavy metal and by its spontaneous resistant mutant was examined. All experiments on Cd uptake were performed with bacterial cells which were cultivated in the absence of Cd. The cells of the sensitive strain were more effective in the removal process than the resistant ones. At high metal concentration (10μg/ml) the dead cells of this strain (1 mg/ml) were able to remove by sorption 55% of added Cd, while at low concentration (1μg/ml) 60 % of this element was taken up by the same amount of active metabolizing cells by sorption and active transport. The resistant strain removed metal only by sorption. Addition of dead cells to the growth medium had a protective effect against the toxicity of Cd towards sensitive cells. Cadmium sorbed by dead bacterial cells was released (86%) to the medium when added to growing culture of the sensitive strain and could be obtained (70%) as the mobile fraction by extraction with 0.1 MNaNO 3 . Es wurde die Effektivität des Cadmium-Entzuges aus flüssigen Medien durch Pseudomonas -Stämme untersucht, welche zum einen gegenüber diesem Schwermetall anfällig waren bzw. welche, die eine spontane Resistenz zeigten. Die Versuche wurden ausschließlich mit Bakterienzellen durchgeführt, welche in Cd-freien Medien kultiviert wurden. Die Zellen des anfälligen Stammes waren beim Cd-Entzug effektiver als die des resistenten. Bei hohen Metallkonzentrationen (10μg/ml) waren die toten Zellen zum Entzug durch Sorption von 55 % des zugegebenen Cd fähig, während bei geringer Konzentration (1 μg/ml) 60% dieses Elements von der gleichen Menge aktiv metabolisierender Zellen durch Sorption und aktiven Transport aufgenommen wurde. Der resistente Stamm entzieht Cd ausschließlich durch Sorption. Zugaben von toten Zellen zum Wachstums medium bewirkten einen Schutzeffekt gegen die Toxizität des Cd auf anfällige Zellen. Cadmium, einmal von toten Zellen aufgenommen, wurde zu 86% wieder ins Medium freigegeben, wenn diese Zellen einer wachsenden Kultur des sensiblen Stammes zugeführt werden. Dieser wieder freigegebene Cd konnte als mobile Fraktion (70%) mittels Extraktion durch 0,1 MNaNO 3 nachgewiesen werden.

Mutants of the TEM-1 beta-lactamase conferring resistance to ceftazidime.

Spontaneous ceftazidime resistant mutants were obtained from an Escherichia coli K12 J62-2 expressing the TEM-1 beta-lactamase (mutation frequency = 10(-9). These mutants produced beta-lactamases with similar molecular weights, kinetic parameters and isoelectric points (pI) to the beta-lactamases produced by ceftazidime resistant clinical isolates which have recently been identified in this laboratory. Mutant enzyme A focused as a doublet band at pI 5.3 with an additional weak pI 5.4 band. The doublet co-focused with the TEM-E2 beta-lactamase, produced by a ceftazidime resistant Klebsiella oxytoca isolate, which was originally obtained in a Liverpool hospital. Mutant enzyme B had a pI identical to the TEM-E1 beta-lactamase produced by a ceftazidime resistant clinical isolate of E. coli found in Belgium. These results suggest that the two beta-lactamases in the clinical strains may have come from simple mutations of the TEM-1 beta-lactamase gene.

Pathotoxin‐Induced Alteration in Protein Synthesis Associated with Susceptibility of Sorghum to Milo Disease

Symptoms of milo disease of sorghum, Sorghum bicolor (L.) Moench, caused by the fungus, Periconia circinata (Mangin) Sacc., can be elicited selectively in susceptible genotypes by treatment with the host‐specific toxin produced by the fungus. Sorghum lines with diverse genetic backgrounds were examined to determine whether disease susceptibility is correlated with an enhanced rate of synthesis of 16‐kilo dalton proteins induced by the toxin. Test materials included near‐isogenic susceptible and resistant lines of grain sorghum ‘Colby’, susceptible and resistant selections of shattercane, toxin‐sensitive selections from China and Nigeria, and spontaneous resistant mutants of Colby. Protein synthesized in root tips after treatment with the toxin were labeled in vivo with [3H]‐leucine, separated by polyacrylamide gel electrophoresis, and detected by fluorography. Toxin treatment enhanced the rate of synthesis of the 16 kD proteins in all susceptible selections but not in any of the resistant selections. These results establish a correlation between susceptibility of sorghum to P. circinata and an increased rate of synthesis of 16 kD proteins induced by the host‐specific toxin from P. circinata.

Incorporation of Exogenous Purines and Pyrimidines by Methanococcus voltae and Isolation of Analog-Resistant Mutants

Methanococcus voltae incorporated exogenous adenine, guanine, hypoxanthine, and uracil, but not thymine. Growth of M. voltae was also sensitive to purine and pyrimidine analogs. Of the 20 analogs tested, 12 were inhibitory at 1 mg/ml. The most effective inhibitors were purine analogs with endocyclic substitutions. Nucleoside analogs and analogs with exocyclic substitutions or additions were less effective. Four purine analogs, 8-aza-2,6-diaminopurine, 8-azaguanine, 8-azahypoxanthine, and 6-mercaptopurine and one pyrimidine analog, 6-azauracil, were especially toxic. The MICs were 20, 0.5, 2.0, 80, and 10 mug/ml, respectively. Spontaneous resistance mutants were isolated for these five analogs. The MICs for these mutants were 20.5, 8.2, >65, >41, and 20.5 mg/ml, respectively. These concentrations far exceeded the solubilities of the analogs and represented an increase in resistance of at least three orders of magnitude. In addition to demonstrating cross resistance to several of the analogs, four of these mutants lost the ability to incorporate exogenous bases. These appeared to be mutations in the salvage pathways for purines and pyrimidines. In contrast, the mutant resistant to 6-mercaptopurine was not defective in purine uptake. Instead, it degraded 6-mercaptopurine. In the presence or absence of high concentrations of the analogs, the growth rates of the resistant mutants were no less than one-half of the growth rate of the wild type in the absence of the analog. The high level of resistance and rapid growth are very desirable properties for the application of the mutants in genetic experiments.

Cloning of the galactokinase gene (galK) from Streptomyces coelicolor A3(2).

Streptomyces coelicolor A3(2) and Streptomyces lividans 66 strains were shown to be sensitive to the galactose analogue 2-deoxy-D-galactose. Spontaneous resistant mutants were isolated that were Gal- and lacked the enzyme galactokinase. The galK gene (structural gene for galactokinase) from S. coelicolor was cloned into S. lividans using the low copy number vector pIJ922. The resulting plasmid (pMT650), which contained a 14 kb insert, complemented gal mutations in both species. The presence of the galK gene on a 2.8 kb EcoRI fragment was confirmed by expressing it in Escherichia coli where it complemented a well characterized galK mutation.

Selection of enhanced cefotaxime resistance in Enterobacter spp.

Four strains of Enterobacter spp. with different chromosomal beta-lactamase expression (inducible, constitutive or negligible) were grown in broth containing either cefoxitin or cefotaxime, then plated on to agar containing 20 mg cefotaxime per litre to quantitate the cefotaxime-resistant mutants present in the population. Spontaneous resistant mutants were initially isolated from each strain at frequencies of 10(-4) to 10(-5). These high frequencies of spontaneous mutation suggested that more than one type of mutational event could yield cefotaxime resistance. Induction of a high level of beta-lactamase in broth cultures was not in itself sufficient to confer a high level of cefotaxime resistance on the population, and increased resistance following selection of resistant mutants did not necessarily correlate with any significant increase in beta-lactamase activity.

Alterations in penicillin-binding proteins of clinical and laboratory isolates of pathogenic Streptococcus pneumoniae with low levels of penicillin resistance.

Several recent surveys of clinical isolates have indicated that substantial fractions of naturally occurring populations of Streptococcus pneumoniae have undergone a distinct upward move in the required minimal inhibitory concentration (MIC) of benzylpenicillin (from a range of 0.006-0.008 to 0.03-0.05 microgram/ml). Evidence is presented that in clinical pneumococcal isolates, penicillin-binding proteins (PBPs) groups 1 and 2 have a decreased affinity for radioactive benzylpenicillin as compared with penicillin-sensitive isolates from the same locale. Exposure of a penicillin-sensitive type 2 strain (MIC, 0.006 microgram/ml) to sequentially increasing concentrations of penicillin allowed the isolation of spontaneous resistant mutants with stepwise increases in the MIC of penicillin required (0.01-0.02, 0.025-0.05, and 0.1 microgram/ml), and in these laboratory isolates too, PBP groups 1 and 2 showed decreased affinity for labeled benzylpenicillin. DNA from the low-level resistant clinical or laboratory isolates could be used to transform the appropriate levels of penicillin resistance into penicillin-sensitive laboratory isolates. These findings suggest that significant fractions of natural pneumococcal populations may have acquired one or two of the low-level penicillin resistance genes.

Allele-specific suppression of dnaA(Ts) mutations by rpoB mutations in Escherichia coli.

Extragenic suppressor mutations for dnaA (Ts) mutations mapping in the rpoB gene (beta-subunit of RNA polymerase) were isolated by selection of spontaneous rifampicin resistant mutants and screening for temperature resistance. Six rpoB mutations were analysed for suppression of 12 different dnaA (Ts) mutations. The analysis showed that all dnaA(Ts) mutations could be suppressed by some rpoB mutation. All six rpoB mutations showed allele specificity when tested for suppression of 12 dnaA(Ts) mutant strains. The allele specificity was found to correlate with the map position of the dnaA (Ts) alleles.

Use of ureidopenicillins for selection of plasmid vector transformants in Pseudomonas aeruginosa and Pseudomonas putida

Broad-host-range plasmids coding for beta-lactamase were successfully selected after transformation of Pseudomonas strains. Transformants of both Pseudomonas aeruginosa and Pseudomonas putida containing plasmid pRO1614 were isolated in media containing low concentrations of piperacillin. These strains were also susceptible to other ureidopenicillins. Similar selections of transformants with carbenicillin, ampicillin, or ticarcillin required high concentrations of antibiotics and yielded backgrounds of spontaneous resistant mutants.

Genetic studies on the yeast Saccharomycopsis lipolytica Inactivation and mutagenesis

AbstractSpontaneous mutants of Saccharomycopsis lipolytica were selected and partially characterized. Several antibiotics and antimetabolites were used for selection of spontaneous resistant mutants from Saccharomycopsis lipolytica. The frequencies of such mutants were mainly arranged between 1 × 10−7 and 5 × 10−6 mutants per cell. But one class of glucosamine resistant mutants (GAMRA) occurred more frequently. Among the resistant mutants different types of dominant and recessive resistant mutants could be observed.UV light was used for inactivation of cells and induction of mutants from S. lipolytica. Comparing four haploid strains only small differences were detected in sensitivity to UV light. UV light at a dosage of 135 J/m2 was applied to increase the mutant frequencies in three haploid strains. Besides auxotrophic, temperature sensitive and colony morphology mutants, some new mutant types like small colony forming mutants, red‐brown coloured mutants, allylalcohol, glucosamine, 2‐deoxyglucose or nystatin resistant mutants, hitherto not described for S. lipolytica, were isolated and partially characterized.

Characterization of a dicarboximide-fungicide-resistant laboratory isolate ofMonilinia laxa

Monilinia laxa, the incitant of blossom blight in stone fruits in Israel, is sensitive to 5μg/ml of the dicarboximide fungicides vinclozolin and iprodione in the growth medium. When a large number of spores, from an isolate never exposed to these fungicides, was seeded on a medium containing 15 μg/ml iprodione, spontaneous resistant mutants appeared at 10-5 frequency. These mutants showed cross resistance to the dicarboximide fungicides vinclozolin, procymidone, l-(3,5-dichloro-phenyl)-3-propen(2)-pyrrolidin-2,5-dion (Co 4462), l-(3,5-dichlorophenyl)-3-methoxymethyl-pyrrolidin-2,5-dion (Co 6054), and to dicloran. Growth rate on fungicide-free medium was similar to that of the parental sensitive strain but sporulation was much reduced. Growth rate on media supplemented with dicarboximide fungicides decreased gradually with increasing fungicide concentrations. The resistance has been stable for more than one year in the absence of fungicides. Artificial inoculation of cherry, apricot and plum fruits previously treated with 500 μg/ml vinclozolin, iprodione, procymidone or Co 6054, with a resistant strain, resulted in brown rot; similar treatments provided full protection of the fruits against the sensitive strain.

Second-site antibiotic resistance mutations in the ribosomal region of yeast mitochondrial DNA

A large proportion of the spontaneous erythromycin resistant mutants isolated from a strain carrying a previously-induced chloramphenicol resistance mutation at cap3 do not map at ery1, the locus most often associated with mitochondrial erythromycin resistance. Most of the new mutations are also nonallelic at spil, spi2, and other known antibiotic resistance loci within the 21S rRNA gene; they are allelic with each other and define the new locus, ery2. Induced second-site erythromycin resistant mutants from the cap (r3) strain, as well as spontaneous or induced mutants from strains carrying a cap (r) 1 mutation, all tend to map at eryl. The cap (r3) mutation is apparently necessary for the expression of erythromycin resistance resulting from a second mutation at ery2.

Isolierung von Streptothricin-resistenten Mutanten ausEscherichia coli K12, Stamm 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.

Isolierung von Streptothricin‐resistenten Mutanten aus Escherichia coli K12, Stamm A19

AbstractMutants with various levels of resistance to streptothricin were isolated from Escherichia coli K12, strain A19 after mutagenesis with N‐methyl‐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.

Plasmid mediated alterations in composition and structure of envelopes of Escherichia coli B/r

Seven transmissible (Tra +), antibiotic resistance (r) plasmids, which confer sensitivity to the filamentous bacteriophage IKe on an Escherichia coli B/r strain harboring them, have been examined for the changes they evoke in the host cell membranes. The plasmids rR48and rR269, like rRM98 [1], cause a significant reduction in the density of the outer membrane and the virtual elimination of its 36 500 dalton protein. These 2 properties do not appear to be altered when rR45, rR199 or rR205 are the resident plasmids. No changes in the inner membrane proteins are seen in any of these strains. In the case of the rR46-bearing strain, the density of the outer membrane is increased and the level of the 36 500 dalton protein is unaltered; in addition, several changes in both inner and outer membrane proteins are seen. Spontaneous IKe resistant mutants isolated from strains lacking the 36 500 dalton protein are either Tra + or Tra −. Since most of them also lack this protein, the latter is not important to the expression of inter-bacterial gene transfer and IKe sensitivity. On freeze fracture, strains lacking the 36 500 dalton protein cleave almost exclusively within the outer membrane. The plasmidless host and the remaining plasmid-bearing strains show a strong fracture plane within the cytoplasmic membrane. Despite the fact that most of the plasmid-bearing strains used are proficient in effecting interbacterial plasmid transfer, no morphological differences which can be correlated with this function have been observed between their etched cell surfaces and that of the plasmidless host.