Synergistic action of some antibacterial agents in studies on albino mice with experimental plague caused by Fr- phenotype strain of the plague microbe]

Possible use of ciprofloxacin combinations with some other antibiotics such as rifampicin, ampicillin, cefotaxime, doxycycline and amikacin was studied on albino mice with experimental plague caused by the pathogen strain (approximately 1000 LD50) deprived of the ability to produce the capsular antigen, fraction I (Fra- phenotype). The combination of ciprofloxacin with ampicillin or doxycycline had no effect on the increase of the survival rate (t<2) evident of inexpediency of its use in the infection caused by the Fra- strains of the plague microbe. The combination of ciprofloxacin and cefotaxime used in definite doses had some effect (t=2.6). The most significant synergistic effect was observed with the use of ciprofloxacin in combination with amikacin or rifampicin (t>3.3-9.0) which made the combination most promising.     

Ofloxacin efficacy in the prophylaxis and treatment of experimental plague due to antigen complete and defective strains of the pathogen]

Strains of the plague microbe, antigen complete and defective by fraction I and mouse toxin had the same in vitro susceptibility to ofloxacin (MIC 0.08 mg/L). The drug was superior in its activity to pefloxacin and especially nalidixic acid. In the experiments with albino mice (prophylaxis, 5 days) the ofloxacin efficacy was lower when the infection was due to the plague microbe strains deprived of the ability to produce fraction I and mouse toxin, evident from a statistically significant increase of the drug ED50 and a decrease of the animal survival percentage. When used in the doses corresponding to the human average daily doses, ofloxacin provided effective animal protection (80 to 100 per cent survival) after the prophylaxis for 7 days and the treatment of the plague infection irrespective of the strains, complete or antigen changed. However, when the infection is due to the antigen changed strain, ofloxacin should be used in the maximum daily doses at least for 7 days.     

Use of monoclonal antibodies to plague microbe antigens at the early stage of infection in white mice for enhancement of the late streptomycin and doxycycline treatment]

It was demonstrated that use for prophylaxy (after 5 h of infection) or for treatment (after 24 h after infection) of the monoclonal antibodies mixture to specific epitops of capsule antigen (fraction 1), lipopolysacharide, murine toxine can prevent development of plague pathogen at 100 of mice infected by approximately 1000 LD50 Yersinia pestis 231. 5-day course of prophylaxy by monoclonal antibodies provided survival of 50 per cent animals. Subsequent use of fraction 1 antigen for 5 days followed by treatment with streptomycin or doxycycline at 6-7-8-9-10 days after infection with Y. pestis 231 prevented infection manifestation at 80 per cent of animals, etiotropic therapy started at the same period was ineffective. When white mice were infected with Y. pestis 231 Fra-, with deleted ability to produce capsule antigen (fraction 1) 80% level of efficacy can be provided by subsequent administration of antibodies to fraction 1 combinated with lipopolysacharide, murine toxine and streptomycin. Use of monoclonal antibodies followed by doxycycline was ineffective.     

Detoxifying effect of antibiotics and their effectiveness in experimental plague at the stage of explicit intoxication]

The effect of antibiotics such as amikacin, rifampicin, doxycycline, polymyxin B and cefotaxime on the toxins of the plague microbe (lipopolysaccharide + fraction II according to Beiker) was studied in vitro and in vivo. The study on the antibiotic neutralization of plague toxins revealed that only polymyxin had toxin neutralizing capacity which depended on the dose. Investigation of the polymyxin effect at various stages of plague infection showed that when polymyxin in a dose of 1250 units and a mixture of plague toxins in lethal doses were administered simultaneously to albino mice, the positive effect amounted to 100 per cent. When the antibiotic was administered 30 or 60 minutes later, the antibiotic efficacy proved to be lower by 90 or 76.6 per cent, respectively. The intoxication in later periods (in 90-120 minutes) resulted in a decrease in animal survival up to 40-15 per cent. It was demonstrated on the model of the plague infection in albino mice that the use of amikacin, cefotaxime, rifampicin or doxycycline during polymyxin therapy at the stage of marked generalization of the infection provided a significant increase in the animal survival (60 to 80 per cent) as compared to that after the use of the same drugs alone (0 to 20 per cent).     

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.     

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.     

Efficacy of plague prophylaxis with streptomycin, tetracycline, and rifampicin in simultaneous immunization of white mice by resistant EV NRIEG strain]

Tetracycline, doxycycline, streptomycin and rifampicin were used for prophylaxis of experimental plague in albino mice (Yersinia pestis 231, approximately 1000 LD50). The antibiotics were administered 5 hours after the infection for 5 days. Tetracycline and doxycycline provided survival of 60 to 75% of the animals, while the respective figure for streptomycin and rifampicin was 100%, but streptomycin and rifampicin inhibited development of plague immunity evident from a lower protection index (PI) by 3-4 orders. The PI for the tetracyclines lowered by 2 orders. Simultaneous prophylaxis with the tetracyclines and immunization by Y. pestis EV Rifr R(SmTc) (10(6) microbial cells) provided not only higher percentage of the animal survival (80-90%) but also development of sufficient plague immunity: PI of 1.0 x 10(5)--5.0 x 10(5). When the animals were infected with Y. pestis 231 R(SmTc) the use of the tetracyclines failed, whereas the use of doxycycline and simultaneous vaccination by EV Rifr R(SmTc) provided survival of 70-85% of the animals. Successive use of inefficient streptomycin (for 2 days) and efficient rifampicin (for 3 days) provided survival only of 30% of the mice. A similar regimen of the successive use of the inefficient and efficient antibiotics (the total term of 5 days) started simultaneously with immunization by EV Rifr R(SmTc) provided survival of 80% of the animals. The use of combined specific and urgent prophylaxis of plague infection due not only to antibiotic susceptible but also to antibiotic resistant strains of the plague pathogen was shown promising.     

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.     

Chromosomal resistance of plague agent to quinolones]

The nature of increasing chromosomal resistance to quinolones was studied in a model of the plague microbe. Five virulent strains of the natural plague microbe (Y. pestis) were used in the experiment: 363 (1/1479), 231, 2385, 2442 and 2444. The one-stage procedure for isolation of the mutants was applied. It was shown that the frequency of the one-stage mutants resistant to oxalinic acid, pefloxacin and ciprofloxacin amounted to 10(-9)-10(-11) and was 2 to 3 orders of magnitude lower than that of the mutants resistant to nalidixic acid. Two types of the plague microbe mutants resistant to the quinolones were detected: those resistant to the quinolones to the generations (Nalr-phenotype) and those resistant to the representatives of the 3rd generation quinolones (Nals-phenotype). The quinolones were not efficient in the treatment of albino mice with experimental plague caused by the quinolone-resistant forms of the plague microbe.     

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.     

Evaluation of the effectiveness of antibacterial substances in treating an experimental form of bubonic plague in monkeys]

The modelling of glandular plague and selection of the conditions for estimating the efficacy of new antibacterials for the treatment of the infection were performed on hamadryads (baboons). The experiments showed that the average LD50 of the culture of a highly virulent strain of Yersinia pestis on its subcutaneous administration to the animals was 2089 viable microbes. In 18 per cent of the episodes the experimental glandular plague in the animals was complicated by secondary plague pneumonia. Subcutaneous administration of 2 x 10(7) viable microbial cell of the plague pathogen caused acute sepsis and the animal death. The treatment of the experimental glandular plague in the hamadryads demonstrated that new antibacterials such as amikacin, netilmicin, ceftriaxone, cefotaxime, ceftizoxime, doxycycline, rifampicin, ofloxacin and ciprofloxacin were not inferior in their efficacy to streptomycin and tetracycline successfully used in the therapy of patients with plague.     

Effectiveness of cefotaxime in experimental plague infection]

Cefotaxime was shown highly efficient in prophylaxis and treatment of experimental plague infection in albino mice. The in vitro activity of cefotaxime against natural strains of the plague microbe was 32 to 64 times higher than that of cefazolin, cephalothin and cefmetazole. The combined use of cefotaxime with amikacin significantly increased the percentage of the survived albino mice with plague infection as compared to the use of the antibiotics alone.     

Evaluation of outcomes of combined specific prophylaxis with the antibiotic resistant immunogenic plague pathogen strain and emergency prophylaxis with aminoglycosides in the experimental plague model in white mice]

It was shown that aminoglycosides (streptomycin, kanamycin, gentamicin, sisomicin, tobramycin, amikacin) prevented manifestation of postvaccine immunity in albino mice immunized by vaccine strain Yersinia pestis EV. Avirulent strain Y. pestis 363 Monr with chromosome resistance to aminoglycosides of the 1st, 2nd and 3rd generations provided manifestation of antiplague immunity when streptomycin, kanamycin, gentamicin and amikacin were administered for prophylaxis. ED50 achieved 1.0-1.2 x 10(3) CFU and in control group (without treatment) 9.3 x 10(2) CFU. Gentamicin and amikacin were highly effective for experimental plague prophylaxis (90-100% animal survival), but inhibited development of postinfective immunity. Protective index (PI) value was 1.1 x 10(2). It was demonstrated that combination of specific prophylaxis (Y. pestis 363 Monr) and emergency prophylaxis with aminoglycosides in albino mice infected with approximately 1000 LD50 of virulent strain Y. pestis 358 (5 hours after infection) was highly effective and provided protective effect against subsequent infection with plague pathogen. Value of PI was 1.1 x 10(5) and practically did not differ from PI (1.7 x 10(5)) in control group (intact mice, immunized with strains EV [symbol: see text] 363 Monr).     

Effect of subinhibitory concentrations of antibiotics on catalase activity of plague microbes]

It was shown that the presence of subinhibitory concentrations of ampicillin, cefotaxime or gentamicin in the cultivation medium had a marked inhibitory effect on the catalase activity of plague microbe. The effect depended on the characteristic features of plague microbe strains and the incubation temperature. When the cells of a virulent strain of the plague microbe Y. pestis 1300 were cultivated at a temperature of 37 degrees C on a medium containing the subinhibitory concentrations of ampicillin or cefotaxime, the pathogen virulence for albino mice significantly decreased.     

Characteristics of the multiplication of a virulent strain of the plague microbe in Xenopsylla cheopis fleas infected parenterally

Xenopsylla cheopis fleas infected parenterally with the virulent strain of plague microbe of gerbil variant preserved the agent to the end of their lives. In the body cavity the microbes retained their ability for reproduction which was, however, limited. During the first seven days after the infection the number of microbes slightly increased and later became stabilized. Its mean indices (mean g) varied within the limits of 500 to 2000 microbe cells per 1 individual, maximum index rarely exceeded 30 000 microbe cells. Parenteral infection with plague agent did not affect essentially the longevity of fleas.     

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.     

The plague

The plague has been one of the most devastating diseases of human history. Despite major advances in diagnosis, prevention, and treatment, it has not been possible to eradicate this infection. Plague is still active in Africa, in Asia and in the Americas, and is classified as a currently re-emerging disease. The plague is mainly a disease of rodents, which is transmitted by fleabites. Humans develop two main clinical forms: bubonic plague (following bites of infected fleas, lethal in 50-70% of the cases in less than a week if an appropriate treatment is not started rapidly), and pneumonic plague (after inhalation of infected droplets, lethal in less than three days in 100% of cases without immediate treatment). Y. pestis, the causative agent of plague, is usually sensitive to most antibiotics, but the first multi-resistant strain was recently described. No efficient and safe vaccines are currently available. The plague bacillus is one of the few organisms that could be used for biological warfare.     

Effectiveness of phosphomycin and its combinations with amikacin and cefotaxime in experimental plague infection]

When administered intramuscularly in doses of 8 and 16 mg/mouse, phosphomycin was highly active in the treatment of albino mice with experimental plague infection (80-100-percent protection of the animals from death). Combinations of phosphomycin with cefotaxime in inefficient or not sufficiently efficient doses had a synergistic effect. When the albino mice were treated with combinations of phosphomycin and amikacin, the percentage of the survived animals significantly increased in comparison to that after the use of the antibiotics alone.     

Prophylactic use of ceftriaxone in combination with F I antigen immunization in studies on uninbred albino mice infected by Yersinia pestis. Antiplague immunity development]

Administration of highly immunogenic (ED50 12.6 mcg/mouse) F I antigen (100 mcg/mouse) to albino mice 5 hours after their contamination approximately with 1000 LD50 of Yersinia pestis 231 provided 99-percent survival of same animals (17-50%) and 2-5-day prolongation of the life-span, that was indicative of the phenomenon analogous to the survival phenomenon observed in infected animals immunized by immunogenic strains of the plague microbe. The experiment on the mice confirmed high efficacy of ceftriaxone (100-percent survival) when used prophylactically for 5 days 5 hours after the contamination by Y. pestis 231 (approximately 1000 LD50) in the dose equivalent to the daily dose for humans. However, no antiplague immunity developed in the survivors: the immunity index (II) of 1.5x10. The use of ceftriaxone according to the same scheme simultaneously with single immunization by F I antigen in a dose of 100 mcg/mouse resulted not only in 100-percent survival of the animals but also in development of expressing antiplague immunity (II 2.2x10(5)). The protection level corresponded to the control with the same live-stock of the animals after a single immunization in the analogous dose of F I antigen (II 3.2x10(4)) and the ceftriaxone use (II 1.0x10(5)), as well as after immunization of the mice by 10(6) microbial cells of Y. pestis EV NIIEG (II 1.2x10(5)). The results of the study are indicative of the prospective use of subsingle vaccines of the new generation based on F I antigen for combined specific and urgent prophylaxis.     

Experimental evaluation of prospects for the use of beta-lactams in plague infection caused by pathogens with plasmid resistance to penicillins]

High therapeutic efficacies of ceftriaxone, ceftazidime, cefotaxime and azthreonam in the treatment of experimental plague induced by beta-lactamase-producing strains of the plague microbe containing R plasmids RP-1, R57b and R40a were shown to correlate with their in vitro antibacterial activities. The therapeutic efficacy of sulbactam/ampicillin was recorded in the treatment of plague induced by the strain containing R plasmids R57b and R40a (the treatment course of 7 days). However, it was lower when the infection was due to the strain containing plasmid RP-1 (beta-lactamase TEM-2). Cefoperazone was not active in the treatment of experimental plague induced by the strains containing plasmids RP-1 and R57b (beta-lactamases TEM-2 and OXA-3). Ceftriaxone versus the antibiotics tested was considered to be the drug of choice for the etiotropic therapy of plague induced not only by the type strains of the plague microbe but also by its variants with the plasmid pattern resistance to penicillins.