Rapid evaluation of the effectiveness of antibacterial drugs in experimental tularemia

Rapid estimation of the protective effect of antibacterial drugs on Fransiella tularensis for not more than 2 days was shown possible in experiments on albino mice infected with tularemia. High efficacy of aminoglycosides (kanamycin, gentamicin, streptomycin, amikacin, netilmicin, tobramycin, sagamycin, ribostamycin and sisomicin), tetracyclines (tetracycline, doxycycline, minocycline and methacycline), rifampicin, phosphomycin and oxolinic acid was determined with the recommended rapid method. Amoxycillin, ampicillin, piperacillin, carbenicillin, erythromycin, levomycetin, cefradine, cefmetazole, cefatrizine, cefoxitin, cefsulodin and bactrim (biseptol) proved to be inefficient against the tularemia causative agent.     

Detection of persistent resistance to antibacterial drugs in various strains of Francisella tularensis]

Under natural conditions, the Francisella tularensis strains AE-261 and P-13864 capable of forming the persist type of resistance to antibacterial drugs and being the cause of the infection in laboratory animals not responding to monotherapy with antibiotics were detectable. The antibioticograms of strains AE-261 and P-13864 under the in vitro conditions did not differ from those of the other studied strains responding to the antibiotic therapy. The observed phenomenon could be associated with individual peculiarities of the strains and their phenotypic variation in the host. Combinations of aminoglycoside antibiotics (streptomycin, gentamicin and amikacin) with rifampicin were shown to be highly active in the treatment of general forms of the infection due to such strains. The combined therapy of tularemia was also considered promising because of its high efficacy when the treatment was started at late periods as well as because unlike the monotherapy with the aminoglycoside antibiotics it provided complete elimination of the pathogen from the host.     

A rapid method of evaluation of the effectiveness of antibacterial drugs]

Efficacy of antibiotics in the treatment of experimental tularemia was studied comparatively on various biological models. It was shown that the antibiotics which proved active against the tularemia microbe in albino mice when studied by the rapid and routine methods were highly efficient in the treatment and prevention of experimental tularemia in rabbits and baboons (hamadryas). The experiments showed basic possibilities to perform rapid estimation (for at least 2 days) of drug efficacy in experimental glanders and melioidosis in golden hamsters. The rapid method developed by the authors was recommended for the use in primary estimation of the efficacy of new drugs in the treatment of tularemia, glanders and melioidosis.     

Specific effect of attenuated strain of Francisella tularensis on the development of radiation induced lesions in experimental animals and effictiveness of antibacterial therapy for lesions induced by radiation combined with virulemt strain of the bacteria

For study of the effects of whole-body gamma-radiation (1 and 4 Gy) on the response of the body to administration of vaccines and virulent strains of tularemia 206 outbred white mice were used. The results of the study shown that the administration of attenuated bacterial cells in 5 days after exposure to radiation (1 and 4 Gy) caused more severe post-radiation effects and the increase in the number of died animals. The severity of the disease was less if mice were vaccinated in 26 days after irradiation (4 Gy). The treatment of tularemia in irradiated mice twith Riphampicin (daily peroral administration, 5 mg/mouse, duration of treatment--7 days) administered in 4 hours after infection was effective and caused high survival of affected mice. The results show effectiveness of the riphampicin treatment of tularemia in the animals exposed to sublethal dose of radiation.     

Change in phagocytic activity toward the agent of tularemia in highly sensitive animals with mixed infections]

An increase of the ingestive and digestive capacity of neutrophils to the homologous causative agent and tularemia microbe was revealed by the opsonophagocytic test in Microtus arvalis, albino mice and guinea pigs infected with sublethal Yersinia pseudotuberculosis and Salmonella typhimurium doses. In subsequent tularemia infection some of the animals displayed a reduction of the septicemia intensity, prolongation of the disease and elevation of the susceptibility threshold. Period of manifestation of the inhibitory action on tularemia coincided with that of the increase in phagocytic activity     

Cefepime/amikacin in the empirical antibacterial therapy for patients with hemoblastosis of different forms]

The results of the use of cefepime (Maxipime) combination with amikacin vs ceftriaxon combination with amikacin in the treatment of 80 patients with different forms of hemoblastosis are presented. Severe infectious complications in the patients were associated with prolonged and deep neutropenia during inductive or antirelapsing chemotherapy. All the patients in the trial were from the group of high risk of infectious complications with the blood neutrophil count under 100 cells/microliter. The duration of neutropenia averaged 12 days (7 to 15). The average period of the treatment with cefepime and amikacin equaled to 13 days (8 to 16). The treatment with cefepime + amikacin was successful in 38 out of 40 patients (95%). The average period of the treatment with ceftriaxon and amikacin equaled to 14 days (7 to 18). The efficacy of the treatment with ceftriaxon + amikacin was 60% (24 patients out of 40).     

Results of the study of bird droppings collected in the Lithuanian SSR for the purpose of searching out tularemia epizootics

There are practically no records of cases of tularemia among humans in the Lithuanian SSR. Nevertheless, the mass sero-allergic survey of the population for tularemia, carried out 10-12 years ago, showed that 2.3% of the adult population in the Republic had had contacts with the causative agent of this infection. The work was aimed at the determination of the present activity of the foci of tularemia. During 6 years in 22 rural districts 2582 samples of avian excrements, containing bones and wool of small animals, were collected and studied by means of the antibody neutralization test (ANT). In 132 (5.1 +/- 0.4%) excrement samples collected on the territory of 12 districts Francisella tularensis antigen was detected. The average ANT titer was 45.2, the maximum titer (10 excrement samples) reached 1: 160. The study revealed the existence of the natural foci of tularemia in Lithuania at present, but their activity proved to be low. The most unfavorable situation was found to exist in western districts of the Republic.     

Effectiveness of the tularemia-antibody erythrocyte diagnostic agent for the detection of specific antigen and antibodies to it]

The authors demonstrated the efficacy of utilization of tularemia antibody erythrocytic diagnostic agent. Data are presented indicating a strict specificity and a high sensitivity of this diagnostic agent for detection of the Vi-antigen both in the tularemia microbe cultures and in the suspensions of the organs of rodents which perished of tularemia, irrespective of the state of cadaver. There was revealed relationship between the sensitivity of the diagnostic agent and the virulence of the tularemia microbe cultures. A possibility of using the diagnostic agent in the antigen neutralization test for detection of specific antibodies in the sera of patients, who sustained the disease, and of the vaccinated humans and animals was revealed.     

Rise in the epizootic activity of a natural focus of steppe-type tularemia in Stavropol Territory and its epidemic consequences

One of recurrent autumn and winter rises in the epizootic activity of the steppe-type focus of tularemia in the Stavropol Territory is described. During the tularemia epizooty of 1981-1982 the population of common voles (Microtus arvalis) was very numerous, while the population of house mice (Mus musculus) was comparatively scarce. Francisella tularensis strains were isolated from different species of rodents (Microtus arvalis, Mus musculus, Apodemus sylvanicus, Rattus norvegicus, Lepus, etc.), from fleas and ticks, as well as from environmental objects (well-water, hay). 38 cases of the disease among nonvaccinated persons, mainly in rural areas, were registered. Anginous-bubonic tularemia was the dominant form of the disease. A complex of measures aimed at the detection of tularemia and its liquidation among the population was carried out.     

Isolation of pathogens other than Yersinia pestis during plague investigations.

From 1975 to 1978, 37 isolates of Pasteurella multocida, 1 of Salmonella enteriditis, and 5 of Francisella tularensis were recovered from 42 mammalian specimens and 1 flea pool submitted for examination for evidence of infection with Yersinia pestis. Most of the specimens were collected during investigations of either a human plague infection or a reported epizootic among rodent populations. All specimens were of species regularly or occasionally involved in plague or tularemia cycles in nature and most were collected in areas of known plague or tularemia activity.     

Low dose vaccination with attenuated Francisella tularensis strain SchuS4 mutants protects against tularemia independent of the route of vaccination

Tularemia, caused by the gram-negative bacterium Francisella tularensis, is a severe, sometimes fatal disease. Interest in tularemia has increased over the last decade due to its history as a biological weapon. In particular, development of novel vaccines directed at protecting against pneumonic tularemia has been an important goal. Previous work has demonstrated that, when delivered at very high inoculums, administration of live, highly attenuated strains of virulent F. tularensis can protect against tularemia. However, lower vaccinating inoculums did not offer similar immunity. One concern of using live vaccines is that the host may develop mild tularemia in response to infection and use of high inoculums may contribute to this issue. Thus, generation of a live vaccine that can efficiently protect against tularemia when delivered in low numbers, e.g. <100 organisms, may address this concern. Herein we describe the ability of three defined, attenuated mutants of F. tularensis SchuS4, deleted for FTT0369c, FTT1676, or FTT0369c and FTT1676, respectively, to engender protective immunity against tularemia when delivered at concentrations of approximately 50 or fewer bacteria. Attenuated strains for use as vaccines were selected by their inability to efficiently replicate in macrophages in vitro and impaired replication and dissemination in vivo. Although all strains were defective for replication in vitro within macrophages, protective efficacy of each attenuated mutant was correlated with their ability to modestly replicate and disseminate in the host. Finally, we demonstrate the parenteral vaccination with these strains offered superior protection against pneumonic tularemia than intranasal vaccination. Together our data provides proof of principle that low dose attenuated vaccines may be a viable goal in development of novel vaccines directed against tularemia.     

Localization of an Amikacin 3′-Phosphotransferase in Escherichia coli

A plasmid-encoded enzyme reported by us to phosphorylate amikacin in a laboratory strain of Escherichia coli has been localized in the bacterial cell. More than 88% of this amikacin phosphotransferase (APH) activity was retained in spheroplasts formed by ethylenediaminetetraacetate-lysozyme treatment of an APH-containing E. coli transconguant known to form spheroplasts readily. By comparison, the spheroplasts retained 94% of deoxyribonucleic acid polymerase I and 98% of glutamyl-transfer ribonucleic acid synthetase, two internal markers, whereas less than 10% of the activity of a periplasmic marker, acid phosphatase, was present in spheroplasts. Treatment of whole cells of the transconjugant with chemical probes incapable of crossing the plasma membrane obliterated acid phosphatase activity, whereas the internal markers deoxyribonucleic acid polymerase I, glutamyl-transfer ribonucleic acid synthetase, and β-galactosidase were virtually unaffected after treatment for 5 min; more than 60% of the APH activity remained. As a control, similar chemical treatment of sonic extracts, in which enzymes were not protected by bacterial compartmentalization, produced more extensive reduction in the activities of all test enzymes, including APH. Spheroplasts preincubated with adenosine triphosphatase were shown by thin-layer chromatography to phosphorylate amikacin. Spheroplasts of cells grown in the presence of H₃³²PO₄ were shown to utilize internally generated adenosine 5′-triphosphate in the phosphorylation of amikacin. The absence of ³²P-phosphorylated amikacin after incubation of [γ-³²P]adenosine 5′-triphosphate with spheroplasts confirmed that exogenous adenosine 5′-triphosphate was not used in the reaction. These results suggest an internal location for APH. This conclusion has implications for the role of such enzymes in aminoglycoside resistance of gram-negative bacteria.     

Tularemia study in Khmel'nitski Province

The differentiated approach to carrying out the vaccinal prophylaxis of tularemia is necessary in view of the determination of the types of the natural foci of this infection and their epizootic manifestation, as well as taking into account concrete local conditions and social factors. At the same time, the level of zooparasitological investigations must be maintained for the timely determination of the activity and spread of the natural foci of tularemia.     

Modeling inhalational tularemia: deliberate release and public health response

Two epidemic modeling studies of inhalational tularemia were identified in the published literature, both demonstrating the high number of potential casualties that could result from a deliberate aerosolized release of the causative agent in an urban setting. However, neither study analyzed the natural history of inhalational tularemia nor modeled the relative merits of different mitigation strategies. We first analyzed publicly available human/primate experimental data and reports of naturally acquired inhalational tularemia cases to better understand the epidemiology of the disease. We then simulated an aerosolized release of the causative agent, using airborne dispersion modeling to demonstrate the potential number of casualties and the extent of their spatial distribution. Finally, we developed a public health intervention model that compares 2 mitigation strategies: targeting antibiotics at symptomatic individuals with or without mass distribution of antibiotics to potentially infected individuals. An antibiotic stockpile that is sufficient to capture all areas where symptomatic individuals were infected is likely to save more lives than treating symptomatic individuals alone, providing antibiotics can be distributed rapidly and their uptake is high. However, with smaller stockpiles, a strategy of treating symptomatic individuals alone is likely to save many more lives than additional mass distribution of antibiotics to potentially infected individuals. The spatial distribution of symptomatic individuals is unlikely to coincide exactly with the path of the dispersion cloud if such individuals are infected near their work locations but then seek treatment close to their homes. The optimal mitigation strategy will depend critically on the size of the release relative to the stockpile level and the effectiveness of treatment relative to the speed at which antibiotics can be distributed.     

Delivery of Aerosolized Liposomal Amikacin as a Novel Approach for the Treatment of Nontuberculous Mycobacteria in an Experimental Model of Pulmonary Infection

Pulmonary infections caused by nontuberculous mycobacteria (NTM) are an increasing problem in individuals with chronic lung conditions and current therapies are lacking. We investigated the activity of liposomal amikacin for inhalation (LAI) against NTM in vitro as well as in a murine model of respiratory infection. Macrophage monolayers were infected with three strains of Mycobacterium avium, two strains of Mycobacterium abscessus, and exposed to LAI or free amikacin for 4 days before enumerating bacterial survival. Respiratory infection was established in mice by intranasal inoculation with M. avium and allowing three weeks for the infection to progress. Three different regimens of inhaled LAI were compared to inhaled saline and parenterally administered free amikacin over a 28 day period. Bacteria recovered from the mice were analyzed for acquired resistance to amikacin. In vitro, liposomal amikacin for inhalation was more effective than free amikacin in eliminating both intracellular M. avium and M. abscessus. In vivo, inhaled LAI demonstrated similar effectiveness to a ∼25% higher total dose of parenterally administered amikacin at reducing M. avium in the lungs when compared to inhaled saline. Additionally, there was no acquired resistance to amikacin observed after the treatment regimen. The data suggest that LAI has the potential to be an effective therapy against NTM respiratory infections in humans.     

Murein degradation inEscherichia coli infected with bacteriophage ϕX174

LY 127935 (moxalactam), a new 1-oxa cephalosporin, was evaluated in vitro in agar dilution testing against 177 different clinical isolates of cephalothin-resistant Enterobacteriaceae andPseudomonas aeruginosa in parallel with amikacin, cephalothin, cefoxitin, and cefamandole. Ninety percent of the isolates were also gentamicin-resistant by disk testing. LY 127935 showed a very high degree of activity against cephalothin-resistant organisms but amikacin was more active in vitro, particularly againstP. aeruginosa. Cefoxitin and cefamandole were consistently less active than either LY 127935 or amikacin.     

Endometrial levels of amikacin in the mare after intrauterine infusion of amikacin sulfate

Amikacin sulfate,^e a semi-synthetic aminoglycoside antibiotic derived from kanamycin, is a broad spectrum antibiotic particularly effective against gram-negative organisms. With this spectrum of activity, amikacin may be an effective treatment for equine metritis.The purpose of this study was to determine the level of amikacin in the endometrium after intrauterine infusion. For three consecutive days, four mares were infused with 2.0 gms once a day and four others were infused with 3.0 gms once a day.Biopsy results indicate that amikacin is readily absorbed into the equine endometrium. The 3.0 gm dose appears to have no therapeutic advantage over the 2.0 gm dose. Amikacin could not be detected in the serum after intrauterine infusion.     

The Use of Fa-Technique for Detecting Francisella Tularensis in Formalin Fixed Material

Tularemia is a highly infectious zoonosis, in Sweden usually occurring in the varying hare. The risk of human infections when diagnosing this disease in the laboratory is high. Therefore, a method for diagnosing tularemia in formalin fixed material with the FA-technique has been tested. 46 necropsied varying hares were examined using this method. 28 of the examined animals were diagnosed with tularemia based on conventional post mortem, histological and bacteriological examinations. 96 per cent of them were positive to the FA-technique test. 18 other animals infected with different bacteria and Toxoplasma were also tested. All the controls were negative.     

1-N-(S-3-amino-2-hydroxypropionyl) gentamicin B (Sch 21420): A collaborative in vitro susceptibility comparison with amikacin and gentamicin against 12,984 clinical bacterial isolates

Schering Compound Sch 21420 is an aminoglycoside with antibacterial activity similar to amikacin but with a potential for renal toxicity lower than that of gentamicin. The in vitro activity of Sch 21420, amikacin, and gentamicin was compared with 12,984 bacterial isolates from six clinical laboratories. Agar dilution and broth microdilution techniques were both used with comparable results. Against most of the Enterobacteriaceae, Sch 21420 had equal or only slightly greater activity than amikacin.Proteus species were generally more susceptible to amikacin and gentamicin. Gentamicin was more active against most isolates included in this study.Pseudomonas aeruginosa recovered in the different institutions showed marked variations in susceptibility to gentamicin; amikacin and Sch 21420 were effective against most gentamicin-resistant strains that produce inactivating enzymes: but all three drugs were ineffective against permeability mutants. If diminished renal- and ototoxicity are confirmed, Sch 21420 promises to be a welcome addition to the antibiotic armamentarium.     

Cytopathic effect of the tularemia microbe on a culture of peritoneal macrophages

Morphological analysis of the process of interaction of tularemia microbe strains differing by virulence with macrophages demonstrated that all these strains produced a lethal effect on macrophages obtained from the animales sensitive to the infection. The macrophages obtained from the animals were but little sensitive to tularemia and were resistant to the action of the causative agent of this infection. The data obtained led to a supposition on the presence in the tularemia causative agent of a factor responsible for its lethal action on the macrophages.