Lack of levofloxacin and moxyfloxacin efficacy in experimental plague of albino mice infected with nalidixic acid resistant pathogen (Nal(r))

The efficacy of levofloxacin and moxyfloxacin vs. the previously tested fluoroquinolones was studied on albino mice with experimental plague due to the Nal(r) mutants of Yersinia pestis 231 and 231 FI-. The plague microbe mutants resistant to nalidixic acid (Nal(r)) generated at a frequency of 10(-10)-10(-9). The resistance to nalidixic acid was not accompanied by the strains loss of the virulence. The Nal(r) mutants were cross resistant to fluoroquinolones (ciprofloxacin, moxyfloxacin). The LD50 for the nontreated animals did not differ from that for the mice treated with nalidixic acid and the fluoroquinolones (when the animals were infected with Nal(r) mutants). The results showed that the criteria of the plague microbe susceptibility/resistance to fluoroquinolones should be revised.     

Thymidine phosphorylase activity and its relationship to trimethoprim in initial strains and thymidine-, thymine-dependent and trimethoprim-resistant mutants of plague microbe]

The comparative study revealed thymidine phosphorylase activity in the initial strains of a plague microbe of the field variety and in thymidine-, thymine-dependent and trimethoprim-resistant mutants of the plague microbe of other varieties. The data fully conformed to the results of the microbiological investigation of the strains' ability to grow on the nutrient media with trimethoprim in the presence of thymine and thymidine. On the basis of these results it appeared possible to divide the initial and mutant strains of the plague microbe into four arbitrary groups: initial strains of the plague microbe of all the varieties except the field ones sensitive to trimethoprim under any temperature conditions of incubation on any medium with any supplements; initial strains of the plague microbe of the field variety resistant to trimethoprim at 28 degrees C in the presence of thymine or thymidine alone; Tmpr mutants whose resistance to trimethoprim at 28 degrees C did not depend on the presence of thymine or thymidine, purine and vitamins, but depended on the presence of these substances at a temperature of 37 degrees C.     

Formation of virulent antigen-modified mutants (Fra-, Fra-Tox-) of plague bacteria resistant to rifampicin and quinolones]

Experiments were performed with two strains of plague bacteria--231 (isolated from marmot) and 358 (isolated from human) and their isogenic variants with Fra- and Fra-Tox- phenotype. Mutants resistant to rifampicin (Rifr) and nalidixic acid (Nalr) appeared independently of pathogen phenotype and genotype with frequency n.10(-8)-n.10(-9), subsequently. Rifr mutation influenced on virulence manifestation at albino mice and antigendeficient variants with Fra- and Fra-Tox- phenotype. In every group of strains highly virulent subcultures were registered. Resistance to nalidixic acid mainly was not associated with virulence loss. Nalr mutants of parent and antigenmodified mutants were cross resistant to fluoroqinolones (ciprofloxacin, ofloxacin, pefloxacin, lomefloxacin). LD50 for untreated albino mice did not differ from LD50, for mice treated with rifampicin (when mice were infected with strain resistant to rifampicin) or with nalidixic acid and fluoroquinolones (when animals were infected with Nalr mutants). Antigenmodified strains of plague bacteria and their Rifr, Nalr mutants were able to overcome specific immune reaction. The drugs should be used in synergic combinations (with aminoglycosides or cephalosporines of III generation) to prevent appearance of virulent strains resistant to rifampicin and fluroquinolones.     

Experimental resistance of Vibrio cholerae el tor to nalidixic acid and fluoroquinolones]

It was shown that sensitivity of Vibrio cholerae eltor P-5879 to tetracycline, levomycetin, furazolidone, trimethoprim/sulfamethoxazole, aminoglycosides, beta-lactams, rifampicin, quinolones in vitro correlated with drugs efficacy in the treatment of experimental cholera of albino mice. Mutants of V. cholerae eltor P-5879 Nalr resistant to nalidixic acid (MIC 160-200 mg/l) formed with frequency 10(-9)-110(-8) had no cross resistance to fluoroquinolones. But the efficacy of ofloxacin, lomefloxacin, norfloxacin against these mutants in vivo reduced, though it was not changed in vitro. Mutants of V. cholerae eltor P-5879 resistant to fluoroquinolones and selected after culturing in the presence of the drugs had cross resistance to all quinolones studied. Infection caused by Cpfr mutant could not be treated with nalidixic acid and fluoroquinolones, therapeutic efficacy of rifampicin and beta-lactams, also reduced though sensitivity in vitro was not changed. The results of investigation proves the necessity of quinolones use for cholerae treatment as it is recommended for other severe enteric infections.     

Isepamycin in prophylaxis and treatment of experimental plague due to FI+ and FI- variants of plague microbe]

The efficacy of isepamycin vs. other aminoglycosides was studied in vitro and on albino mice with experimental plague due to natural antigen valuable strains of the plague microbe and the pathogen variants deprived of the ability to produce the capsular antigen fraction I (FI- phenotype). The MICs of isepamycin for the strains of the plague microbe (20 FI+ and 20FI-) were 1.0-4.0 mg\l, that did not differ from those of streptomycin, kanamycin, amikacin and tobramycin. The ED50 of isepamycin in the prophylaxis and treatment of the experimental plague of the mice had no statistically significant differences from the ED50 of the other aminoglycosides. The efficacy index of isepamycin was > 10(4), that did not differ from that of streptomycin, amikacin and gentamicin, irrespective of the strain phenotype (Y. pestis 231 FI+ or Y. pestis 231 FI-). The same as the other aminoglycosides, isepamycin in doses equivalent to the human average daily doses, protected 80-100% of the albino mice from death when used in the prophylaxis and therapy of plague irrespective of the strain phenotype. The results of the study made it possible to consider isepamycin as an agent promising for the prophylaxis and treatment of plague.     

Doxycycline in the prevention of experimental plague induced by plague microbe variants]

A comparative study was performed on the efficacy of doxycycline in experimental plague infection induced in albino mice by strain 231 of the plague microbe and its variant 231 Fra- deprived of the ability to produce the fraction I antigen. It was shown that the LD50 for strain 231 during animal treatment with doxycycline was significantly higher than that for variant 231 Fra-. Prophylaxis of the plague infection caused by the Fra- forms of the plague microbe required significantly higher doses of doxycycline (ED50) than that of the infection caused by the Fra+ forms. The use of the daily maximum permissible doses of doxycycline (50 to 100 mg/kg a day) for 10 days in treatment of albino mice infected with the strain Fra- did not provide animal survival at the level higher than 60 to 70 per cent while the survival rate in the animals infected with the strain Fra+ of the plague microbe and treated according to the same scheme amounted to 90-100 per cent. The lower therapeutic efficacy of doxycycline in the treatment of the infection caused by the fractionless variant of the plague microbe should be considered in development of rational schemes for prophylaxis and treatment of plague.     

Antibiotic sensitivity of plague microbe strains from foreign countries]

The method of serial dilutions on the Hottinger agar was applied to comparative assay of antibiotic sensitivity in 50 strains of the plague microbe isolated abroad and in 5 strains isolated in the plague focus in the Central Caucasus. The antibiotics used in the assay were the following: streptomycin, gentamicin, doxycycline, monomycin, kanamycin, tetracycline, erythromycin, ristomycin, lincomycin and polymyxin M. Irrespective of the origin, all the isolates were resistant to erythromycin, lincomycin and polymyxin M. The levels of the sensitivity to the other antibiotics were different. The data serve as a ground for the statement that there is no tendency to development of antibiotic resistance in the plague microbe in patients treated with high doses of the antibiotics and mainly streptomycin. Along with streptomycin, such antibiotics as gentamicin, tetracycline, doxycycline and kanamycin are useful in the therapy of plague and require further investigation.     

Spheroplasts of plague microbe strains from the Transcaucasian uplands and their capacity for pesticin synthesis]

Spherical formations of the plague microbe strains from the Transcaucasian Upland, I plague microbe strain of the sandwort variety and I strain of the marmot variety were obtained under the effect of lithium chloride. They had the remains of the cell wall, were viable, sensitive to osmotic shock, preserved sensitivity to the specific bacteriophage and pesticins. All this was evident of isolation of the spheroplasts of the plague microbe. The spheroplasts showed a capacity for pesticin production. The pesticin synthesis by the spheroplasts of the plague causative agent from the Transcaucasian Upland increased with an increase in the content of lithium chloride in the medium. The largest inhibiiton zones were observed, when 0.7-0.8 per cent of lithium chloride were present in the medium. In the spheroplasts of the plague causative agent from the Mountain Altai (the marmot variety) the pesticin synthesis was inhibited with an increase in the content of lithium chloride in the medium. The activity spectrum of the pesticins of the spheroplasts of the plague causative agent from the Transcaucasian Upland and the spheroplasts of the strains of the marmot and sandwort varieties was broader than that of the rod-like forms of these strains. The indicator properties were found in the strains of the plague microbe of the marmot and sandwort varieties with respect to the pesticins of the spheroplasts of the sel-like producing organisms and organisms from the Transcaucasian Upland.     

Point mutations in the DNA polymerase gene of human cytomegalovirus that result in resistance to antiviral agents.

Three independently isolated mutants of human cytomegalovirus strain AD169 were found to be resistant to ganciclovir at a 50% effective dose of 200 microM. Phosphorylation of ganciclovir was reduced 10-fold in mutant-infected cells compared with AD169-infected cells. All three mutants were also determined to be resistant to the nucleotide analogs (S)-1-[(3-hydroxy-2- phosphonylmethoxy)propyl]adenine (HPMPA) and (S)-1-[(3-hydroxy-2-phosphonylmethoxy)propyl]cytosine (HPMPC) and hypersensitive to thymine-1-D-arabinofuranoside (AraT). Single base changes resulting in amino acid substitutions were demonstrated in the nucleotide sequence of the DNA polymerase gene of each mutant. The polymerase mutation contained in one of the mutants was transferred to the wild-type AD169 background. Ganciclovir phosphorylation in cells infected with the recombinant virus produced by this transfer was found to be equivalent to that of AD169-infected cells. The ganciclovir resistance of the recombinant was reduced fourfold compared with that of the parental mutant; however, the recombinant remained resistant to HPMPA and HPMPC and hypersensitive to AraT. The ganciclovir resistance of the mutants therefore appears to result from mutations in two genes: (i) a kinase which phosphorylates ganciclovir and (ii) the viral DNA polymerase.     

New Escherichia coli gyrA and gyrB mutations which have a graded effect on DNA supercoiling

Summary We isolated new gyrA and gyrB mutations in Escherichia coli which have a graded effect on DNA supercoiling. The mutants, selected respectively for resistance to nalidixic acid and coumermycin, were sorted by means of a rapid in vivo assay of DNA gyrase activity (Aleixandre and Blanco 1987). Cells carrying a gyrB (Cou^r) mutation usually showed a decrease in DNA supercoiling, which would indicate a reduction in gyrase activity. In contrast, most of the gyrA (Nal^r) mutations had no significant effect on DNA supercoiling. Moreover, they conferred a high level of resistance to nalidixic acid and other quinolones, thus being similar to the gyrA(Nal^r) mutants currently used. We also detected rare gyrA mutants showing a reduction in DNA gyrase activity. These mutants were, in addition, resistant to only low concentrations of quinolones, which allowed us to use the phenotype of partial quinolone resistance as an indicator to score gyrA mutations affecting DNA supercoiling. When gyrB mutations were introduced into the gyrA mutants, these became more sensitive to quinolones and a decrease in supercoiling was observed. Moreover, the topA10 mutation sensitized gyrA(Nal^r) cells to quinolones. We conclude therefore that the GyrA-dependent quinolone resistance is diminished as a consequence of the reduction either in topoisomerase I or gyrase activities.     

Assessing the Risk That Phytophthora melonis Can Develop a Point Mutation (V1109L) in CesA3 Conferring Resistance to Carboxylic Acid Amide Fungicides

The risk that the plant pathogen Phytophthora melonis develops resistance to carboxylic acid amide (CAA) fungicides was determined by measuring baseline sensitivities of field isolates, generating resistant mutants, and measuring the fitness of the resistant mutants. The baseline sensitivities of 80 isolates to flumorph, dimethomorph and iprovalicarb were described by unimodal curves, with mean EC(50) values of 0.986 (±0.245), 0.284 (±0.060) and 0.327 (±0.068) μg/ml, respectively. Seven isolates with different genetic background (as indicated by RAPD markers) were selected to generate CAA-resistance. Fifty-five resistant mutants were obtained from three out of seven isolates by spontaneous selection and UV-mutagenesis with frequencies of 1×10(-7) and 1×10(-6), respectively. CAA-resistance was stable for all mutants. The resistance factors of these mutants ranged from 7 to 601. The compound fitness index (CFI = mycelial growth × zoospore production × pathogenicity) was often lower for the CAA-resistant isolates than for wild-type isolates, suggesting that the risk of P. melonis developing resistance to CAA fungicides is low to moderate. Among the CAA-resistant isolates, a negative correlation between EC(50) values was found for iprovalicarb vs. flumorph and for iprovalicarb vs. dimethomorph. Comparison of the full-length cellulose synthase 3 (CesA3) between wild-type and CAA-resistant isolates revealed only one point mutation at codon position 1109: a valine residue (codon GTG in wild-type isolates) was converted to leucine (codon CTG in resistant mutants). This represents a novel point mutation with respect to mutations in CesA3 conferring resistance to CAA fungicides. Based on this mutation, an efficient allelic-specific PCR (AS-PCR) method was developed for rapid detection of CAA-resistance in P. melonis populations.     

Sympatric speciation of the plague microbe <Emphasis Type="Italic">Yersinia pestis</Emphasis>: Monohostal specialization in the host–parasite marmot–flea (<Emphasis Type="Italic">Marmota sibirica–Oropsylla silantiewi</Emphasis>) system

An ecological scenario of the origin of the plague microbe that is interpreted in the light of modern Darwinism (synthetic theory of evolution) is presented. It is shown that the plague microbe emerged from a clone of the psychrophilic saprozoonotic pseudotuberculosis microbe Yersinia pseudotuberculosis O:1b in the mountain steppe landscapes of Central Asia in the Sartan time, 22000–15000 years ago, in the monohostal Mongolian marmot (Marmota sibirica)–flea (Oropsylla silantiewi) host–parasite system. It was noted that the evolutionary process described corresponds to the sympatric form of speciation by transition of the clone of migrant founders to a new, already-existing ecological niche. It was established that monohostal specialization of the plague microbe was made possible due to heterothermia (5–37°C) of marmots in the hibernation period. The factors of the speciation process—isolation, the struggle for existence, and natural selection—were analyzed.     

Exploration of natural foci of tularemia and plague in Armenia using the serological examination of bird droppings and excrements of predatory mammals]

Forty strains of tularemia and 619 of plague microbes were isolated in 1974 in bacteriological examination of natural plague and tularemia foci from a great number of small mammals and their ectoparasites. At the same time in serological examination (in the antibody neutralization test) of bird pellets, 52 mummified cadavers, and 34 excretion samples of mammalian beasts of prey collected in Armenia (its central and North-Western part) in 1973 the antigen of tularemia microbe was revealed in 73, 8, and 3, and of plagye--in 42, 5, and 1 cases, respectively. In one of the sites examined the number of positive findings failed to exceed 10--17%, this indicating a low intensity of the epizootic in the majority of the places. Judging by the mean titres of the serological test, which varied from 1:12 to 1:1428 in examination for tularemia and from 1:12 to 1428 in examination for plague, it was possible to detect both epizootics coursing during the examination, and those which occurred several monts ago. Tularemia and plague foci were not infrequently present at the same territories, and these diseases could involve the same individual animals of Microtus arvalis (Pall.). The data obtained pointed to the greater effectiveness of examination of bird's pellets and excretions of mammalian beasts of prey for the reconnaisance investigation of the natural foci of plague and tularemia in comparison with the bacteriological methods applied usually.     

Antibiotics of the aminoglycoside group (gentamicin, sisomicin and amikacin) in the prevention and treatment of experimental plague]

Activity of aminoglycosides such as gentamicin, sisomicin and amikacin against plague microbe strains of natural origin was studied in vitro. It was also studied in prophylaxis and treatment of experimental plague infection in albino mice. The MAC of gentamicin and sisomicin for 50 strains of the plague microbe was 0.2-1.6 micrograms/ml. For the majority of the strains it was 0.4 micrograms/ml. The amikacin MICs were 0.4-3.2 and 0.8 micrograms/ml, respectively. High efficacy of gentamicin, sisomicin and amikacin was shown in prophylaxis and treatment of experimental plague infection in albino mice. The optimal doses of the antibiotics were determined. Under definite conditions such as the use of short-term regimens and higher intervals, advantages of sisomicin over gentamicin and amikacin in prophylaxis of experimental plague infection were observed.     

Role of the outer membrane in the accumulation of quinolones by Serratia marcescens

Accumulation of four quinolones by Serratia marcescens was measured fluorometrically. The passage of quinolones through the outer membrane was studied in both lipopolysaccharide-deficient and porin-deficient mutants. The lipopolysaccharide (LPS) layer formed a partially effective barrier for highly hydrophobic quinolones such as nalidixic acid. Quinolones with a low relative hydrophobicity coefficient seemed to pass preferentially through the water-filled Omp3 porin channels. Results were confirmed when Omp3 was cloned in a porin-defective Escherichia coli.     

Origin of the plague microbe <Emphasis Type="Italic">Yersinia pestis</Emphasis>: Structure of the process of speciation

The origin and evolution of the plague microbe Yersinia pestis are considered in the context of propositions of modern Darwinism. It was shown that the plague pathogen diverged from the pseudotuberculous microbe Yersinia pseudotuberculosis O:1b in the mountain steppe landscapes of Central Asia in the Sartan: 22000-15000 years ago. Speciation occurred in the tarbagan (Marmota sibirica)-flea (Oropsylla silantiewi) parasitic system. The structure of the speciation process included six stages: isolation, genetic drift, enhancement of intrapopulational polymorphism, the beginning of pesticin synthesis (genetic conflict and emergence of hiatus), specialization (stabilization of characteristics), and adaptive irradiation (transformation of the monotypic species Y. pestis tarbagani into a polytypic species). The scenario opens up wide prospects for construction of the molecular phylogeny of the plague microbe Y. pestis and for investigation of the biochemical and molecular-genetic aspects of “Darwinian” evolution of pathogens from many other nature-focal infections.     

Escherichia coli females defective in conjugation and in adsorption of a single-stranded deoxyribonucleic acid phage

We predicted that, among mutants resistant to infection by single-stranded deoxyribonucleic acid viruses, there would be some also resistant to "infection" by single-stranded conjugal deoxyribonucleic acid. Approximately 5% of the Escherichia coli K-12 females selected for resistance to phage ST-1 were defective as recipients in conjugation. These spontaneous mutants fell into two classes. Type A accepted both plasmid and chromosomal markers at greatly reduced frequencies (<10(-6) of normal for at least one strain), formed "rough" colonies, and (unlike their parent) were nonflagellated. Type B strains accepted both chromosomal and plasmid markers at reduced frequencies (10(-2) to 10(-1) of normal), were temperature sensitive for growth, and showed increased susceptibility towards antibiotics and deoxycholate. Both classes of mutants also were resistant to certain female-specific viruses.     

Results of using the serological method of epidemiological investigation of plague foci in the Muyunkum and Eastern Kyzylkum deserts in 1978-1982

Results of serological examination of 81,853 mammals from the Muyunkum and 26,303 from the Eastern Kyzylkum deserts were compared with results of a corresponding bacteriological examination for plague. In both foci and in all phases throughout the epizootic of plague, the serological method made it possible to detect infected animals 6-10 times more frequently in comparison with the bacteriological method. The big meriones mice, i.e., the basic carriers of plague in natural foci, were found to be infected considerably more (in percentage) frequently than the meridian meriones. Differences in the reactivity of meriones to the invasion of plague microbe were observed in both foci (the indices of titres in the antigen neutralization reaction were considerably higher in the Eastern Kyzylkums). The serological method of investigation is highly informative in evaluating both the present state of epizootic activity of a plague focus and its activity in the past.