Development of a method for the quantitative assessment of the microorganism sensitivity to antibiotics by using discs. A study of the patterns of doxycycline diffusion from discs into the nutrient agar]

Doxycycline concentrations provided by the antibiotic diffusion from paper discs into sterile agarized medium and the medium plated with staphylococci and Coli bacteria were studied at different distances from the disc center. Infection of the nutrient medium with the test cultures had no effect on the antibiotic diffusion rate. A linear relation between the logarithm of the antibiotic concentration in the agar and distance from the disc centre were found. Probably it is possible to determine the MIC of the antibiotic with respect to various microorganisms by the value of the radius of the growth inhibition zone around the disc using diagrams expressing such a relation.     

Method of determining the antibiotic sensitivity of anaerobic microorganisms using standard disks

Three variants of the procedure for determination of antibiotic sensitivity in anaerobic microorganisms with the use of standard paper discs were developed. According to the first variant the solid nutrient medium is melted at 46 degrees C and mixed with the culture of the microbe being tested. The mixture is added to the cover of a Petri dish. When the medium becomes solid, antibiotic sensitivity discs are placed onto the agar surface. After that one more layer of the medium is added. The medium is allowed to solidify and some more medium is poured near the cover edge. Immediately after that the Petri dish is placed with its flat surface onto the agar layer in its cover. According to the first and second variants the mixture of the medium and culture is added to a Petri dish and immediately a transparent gas-proof polymer film of the dish size is placed onto the agar surface. Previously antibiotic paper discs or solutions are fixed on the films. THe incubation temperature for all three variants is 37 degrees C. The procedure allows one to observe the culture growth and to obtain the results earlier than in case the culture is incubated in an aerostate. The procedure is simple and saves labor and time.     

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.     

Calcium and magnesium in Mueller-Hinton agar and their influence on disk diffusion susceptibility results

Concentrations of calcium and magnesium were determined for 39 lots of media, including broth and agar media used for susceptibility tests and plain agar. In addition, the effect that media with and without physiological levels of these divalent cations would have on the disk diffusion susceptibility of 21 strains ofPseudomonas aeruginosa to four antimicrobics was also ascertained. Mueller-Hinton agar showed a wide variation in calcium and magnesium content. Mueller-Hinton broth contained lower concentrations of calcium and magnesium, and there was little lot-to-lot variation. Lots of Mueller-Hinton agar with higher concentrations of calcium and magnesium yielded smaller zone diamaters than those with lower concentrations. Even at equal cation concentration, zones of inhibition varied from lot to lot. Since the addition of calcium and magnesium to Mueller-Hinton agar to obtain a predetermined level did not result in equivalent zone diameters, performance testing of susceptibility media is recommended.     

Effect of different grades of agar-agar on the nature of the test microbe growth inhibition zones in controlling antibiotic activity by means of agar diffusion]

The effect of various agar grades on the size and margin character of the inhibition growth zones in assay of antibiotic activity by the agar diffusion method was studied. It was shown that not all the agar grades could be used in antibiotic activity assay. Depending on the agar type the size of the inhibition growth zones produced by the same antibiotic concentration significantly varied. The variations in the size of the inhibition growth zones depended on the agar ability to bind antibiotics and were mainly defined by the agar purity. The agars with low content of nitrogen admixtures bound the antibiotics to a low extent. The commerical grades of the agars of the South-Sea and Korsakov Plants, the experimental grade of the TINRO agar with additional purification, as well as the agars imported from Argentina and France proved to be most useful for determination of the antibiotic activity by the agar diffusion method.     

Comparison of Different Agar Diffusion Methods for the Detection of Antimicrobial Residues in Slaughter Animals

The different agar diffusion methods were compared using antibiotic and sulphonamide-impregnated filter-paper discs and the kidneys of healthy and emergency-slaughtered pigs and cows after slaughter. No method used seemed to be sensitive to all antimicrobial drugs preimpregnated onto discs. Tetracycline yielded a greater zone of inhibition at pH 6 than at pH 8 and aminoglycosides, erythromycin, polymyxin B and lin cornycin at pH 8 than at pH 6. It therefore seems necessary to use different pHs (6 and 8). The addition of trimethoprim to the medium is necessary for the detection of sulphonamides. Bacillus subtilis BGA used as the test organism was more sensitive to sulphonamides on the “Test agar for the inhibitor test” containing trimethoprim than on the “Iso-Sensitest agar” also containing trimethoprim. The addition of trimethoprim to “Test agar for the inhibitor test” is recommended at pH 8 but not at pH 6 because false-positive cases (with inhibition zones > 2 mm) were observed at pH 6 with trimethoprim on the kidneys of healthy pigs.     

Antibiotic Susceptibility Patterns of Recent Isolates of Corynebacterium diphtheriae

A variety of factors which might affect zone sizes were studied with strains of Corynebacterium diphtheriae; a standard disc method for antimicrobial sensitivity testing was used. Moderate variations in inoculum size, inoculum preparation, and pH of Mueller Hinton agar (MHA) did not appreciably affect zone sizes. The addition of blood to MHA was necessary to insure the growth of all C. diphtheriae strains on all lots of MHA. Zone diameters on MHA with blood were consistently 4 to 9 mm smaller than on plain MHA; however, zone diameters were within the sensitive range for seven antibiotic discs used on both media. Minimal inhibitory concentration (MIC) values for penicillin, erythromycin, and rifampin were determined by a plate dilution method. The geographical source, toxigenicity, and type of the strains showed no significant correlation with MIC values or zone diameters for eight antibiotic discs. When MIC values were compared to obtainable blood levels, all of the strains appeared to be sensitive with MIC values of 相似文献   

Predilection of Segmental Glomerulosclerosis Lesions for the Glomerulotubular Junction Area in Type 1 Diabetic Patients: A Novel Mapping Method

The location of segmental glomerular lesions in relation to the vascular or tubular pole may have diagnostic or prognostic significance. We have developed a model-based method to estimate the distance from a glomerular lesion to a given landmark (vascular or tubular pole) or the glomerular center and applied this to biopsies from 5 microalbuminuric, 5 normoalbuminuric and 7 proteinuric type 1 diabetic patients and 5 normal controls. The distance from each glomerular adhesion to the glomerulotubular junction was measured and divided by the glomerular radius, allowing comparability among different glomeruli, assuming a spherical shape for Bowman''s capsule, an assumption which was validated. The frequency of adhesions in 6 glomerular zones with equal height (zone I adjacent to the glomerulotubular junction and zones II–VI progressively farther away) was determined: 59% of adhesions were in zone I, 15% in zone II, 16% in zone III, 7% in zone IV and 3% in zone VI (adjacent to the hilus). In glomeruli with only one adhesion, 82% of these were in zone I. This new method accurately localizes segmental lesions within glomeruli and revealed a marked predilection in type 1 diabetic patients for segmental sclerosis to develop at the glomerulotubular junction.     

Multifactorial experiment on the combined action of doxycycline and a low molecular weight immunomodulator of microbial origin in experimental anthrax infection

Chemotherapeutic efficacy of combined therapy of experimental anthrax infection with subtherapeutic doses of doxycycline and a low molecular weight immunomodulator of microbial origin was studied with mathematical design of the experiment and multifactorial analysis. A marked synergistic effect of oral doxycycline and the immunomodulator was observed. The results of the multifactorial experiment were computer processed and polynomial statistic models (the second order equations) describing the survival rate and mean lifespan (MLS) were derived. The equal level lines characterizing the survival rate and MLS were plotted against the fixed values of the time factor of administering the immunomodulator and the dose of the antibiotic. The doses of the immunomodulator and the time of its administration were optimized with respect to the maximum therapeutic effect with doxycycline subtherapeutic doses.     

Simplified, Accurate Method for Antibiotic Assay of Clinical Specimens

Large glass plates are used for this modified agar-well diffusion assay method, allowing up to 81 replications on a single plate. With a specially designed agar punch, it is possible to prepare the small agar wells very quickly. The saving in serum resulting from fewer replications of standards with the large plates, and the small volume of the agar wells, makes it economically feasible to use pooled human serum for the standard antibiotic solutions. Methods are described for preparing the standard solutions, and for providing controls for the deterioration of standards and unknowns. Procedures for preparing and maintaining the commonly used assay organisms are presented. Serum specimens are tested directly rather than diluting them to a narrow range of antibiotic concentrations. This is possible because of a procedure for calculations that recognizes the curvilinear relationship between zone sizes and antibiotic concentrations. Adaptation of this method to a number of the commonly used antibiotics is described. With this method, it has been possible to test large numbers of clinical specimens in a minimal time, and with accuracy consistently better than 10%.     

Interlaboratory variability of disc diffusion and agar dilution susceptibility tests with cefamandole and cephalothin

Reproducibility of antimicrobic susceptibility tests was estimated by examining control data accumulated during a multicenter study for evaluating cefamandole and cephalothin. The precision of agar dilution minimal inhibitory concentrations was compared with the standardized Bauer-Kirby disc method. Regression lines were established for each antimicrobic and were used to calculate the range of minimal inhibitory concentration values that corresponded to the observed ranges in zone sizes, thus permitting a comparison of the two types of procedures. The precision of the disc method was equal to or greater than that of the agar dilution method.     

A rapid immunofluorescent method of determining the antibiotic sensitivity of brucella

Optimal conditions for rapid assay of Brucella antibiotic sensitivity with the immunofluorescent method were developed. With this method high sensitivity of the main Brucella species to tetracycline, doxycycline and rifampicin was confirmed. It was found actually possible to use the immunofluorescent method for rapid assay of Brucella antibiotic sensitivity in practice.     

Accurate assessment of antibiotic susceptibility and screening resistant strains of a bacterial population by linear gradient plate

The dynamics of a bacterial population exposed to the minimum inhibitory concentration (MIC) of an antibiotic is an important issue in pharmacological research. Therefore, a novel antibiotic susceptibility test is urgently needed that can both precisely determine the MIC and accurately select antibiotic-resistant strains from clinical bacterial populations. For this purpose, we developed a method based on Fick's laws of diffusion using agar plates containing a linear gradient of antibiotic. The gradient plate contained two layers. The bottom layer consisted of 15 mL agar containing the appropriate concentration of enrofloxacin and allowed to harden in the form of a wedge with the plate slanted such that the entire bottom was just covered. The upper layer consisted of 15 mL plain nutrient agar added with the plate held in the horizontal position. After allowing vertical diffusion of the drug from the bottom agar layer for 12 h, the enrofloxacin concentration was diluted in proportion to the ratio of the agar layer thicknesses. The uniform linear concentration gradient was verified by measuring the enrofloxacin concentration on the agar surface. When heavy bacterial suspensions were spread on the agar surface and incubated for more than 12 h, only resistant cells were able to form colonies beyond the boundary of confluent growth of susceptible cells. In this way, the true MIC of enrofloxacin was determined. The MICs obtained using this linear gradient plate were consistent with those obtained using conventional antibiotic susceptibility tests. Discrete colonies were then spread onto a gradient plate with higher antibiotic concentrations; the boundary line increased significantly, and gene mutations conferring resistance were identified. This new method enables the rapid identification of resistant strains in the bacterial population. Use of the linear gradient plate can easily identify the precise MIC and reveal the dynamic differentiation of bacteria near the MIC. This method allows the study of genetic and physiological characteristics of individual strains, and may be useful for early warning of antibiotic resistance that may occur after use of certain antimicrobial agents, and guide clinical treatment.     

Multifactor study of combined effects of an antibiotic and polysaccharide in experimental infection]

Multifactorial analysis was applied to the study of the combined effect of doxycycline and a polysaccharide of microbial origin in experimental plague infection. A marked synergistic action of the antibiotic and polysaccharide used in subtherapeutic doses in treatment of the infection was observed. By the results of the experiments polynomial statistic models of the 2nd order were designed and nomographs or equal level lines were plotted. The models and nomographs described the animal survival rate and lifespan within a wide range of the control parameters. The dose/time regimens for the use of the polysaccharide combination with doxycycline were optimized on the basis of the multifactorial analysis.     

Influence of the culture medium on antibiotic susceptibility testing of food-associated lactic acid bacteria with the agar overlay disc diffusion method

AIMS: To investigate the influence of the culture medium on antibiotic susceptibility testing of food-associated lactic acid bacteria (LAB) with the agar overlay disc diffusion (DD) method. METHOD: The antibiotic resistance profile of 39 food-associated lactobacilli and enterococci was determined with the agar overlay DD method using a defined medium (i.e. Iso-sensitest agar; ISA) or an undefined medium (i.e. de Man, Rogosa, Sharpe or MRS agar). RESULTS: The study revealed that ampicillin discs and, although to a lesser extent, also tetracycline discs consistently produced larger zones on MRS medium compared to ISA medium. For the antibiotics gentamicin, bacitracin and erythromycin, the radius of the inhibition zones produced on MRS medium was significantly smaller in relation to ISA. For categorizing LAB isolates into resistant, intermediate and susceptible groups, it was demonstrated that major errors can occur in determining bacitracin and gentamicin resistance if MRS medium instead of ISA medium is used. On the other hand, the performance of both media was found to be equivalent for testing tetracycline resistance. CONCLUSIONS, SIGNIFICANCE AND IMPACT OF THE STUDY: Despite the fact that MRS medium generally supports the growth of lactic acid bacteria much better than the nutrient-poor ISA medium, the present study clearly demonstrates that both media are not compatible in susceptibility testing against various classes of antibiotics. These results may stimulate future discussions on a generally recommended DD method for susceptibility testing of food LAB strains.     

Pharmacokinetic study of implantable gentamycin preparations. I. Antibiotic pharmacokinetics in the implantation area and an evaluation of the prolonged effect of the preparations

Gentamicin pharmacokinetics was studied in the zone of subcutaneous implantation to rats of Septopal, a dosage form based on polymethylmethacrylate stable in vivo (4.5 mg of the antibiotic) and of preparations based on biodegradable polymers such as monocarboxycellulose, alginic acid, nonmodified and modified collagens (1 and 5 mg of the antibiotic). A three-phase pattern of gentamicin level changing in the implantation zone was observed: (1) rapid increasing and decreasing of the antibiotic concentration, (2) stabilization of the gentamicin content at a practically constant level and (3) slow lowering of the antibiotic level in the tissue. Comparison of the areas under the concentration/time curves showed that the modified collagen, alginic acid and monocarboxycellulose had the highest prolongation effect among the biodegradable polymers. Their use in the compositions provided during the first hours after the implantation the antibiotic concentrations in the administration site equal to tens micrograms per 1 g of the tissue. After that during 14 days the concentration of gentamicin in the implantation zone maintained at the level higher than its MIC for the main pathogens of wound infections.     

Randomised double blind trial of single dose doxycycline for treating cholera in adults.

OBJECTIVE--To compare the efficacy of a single dose of doxycycline (200 or 300 mg) with the standard multiple doses of tetracycline in patients with cholera. DESIGN--Randomised double blind controlled trial. Patients were given a single 200 mg dose of doxycycline, a single 300 mg dose of doxycycline, or multiple doses of tetracycline (500 mg, six hourly intervals). SETTING--Hospital in Bangladesh treating diarrhoea. PATIENTS--261 Patients aged over 15 admitted to the hospital with severe dehydration due to acute watery diarrhoea associated with Vibrio cholerae. All vibrios isolated from the stools and rectal swabs of patients, including those patients with prolonged excretion of vibrios, were sensitive to tetracycline. The stools of all patients at admission were negative for shigella and salmonella. INTERVENTIONS--All patients received rapid intravenous acetate solution for the first four hours after admission to hospital. They were then entered in the study and randomised. Oral rehydration was started immediately after the intravenous treatment. If signs of severe dehydration reappeared during oral treatment patients were given rapid intravenous acetate solution until dehydration was fully corrected. MAIN OUTCOME MEASURES--Stool output in first 24 hours and till diarrhoea stopped, total intake of oral rehydration fluid, duration of diarrhoea, and excretion of vibrio after receiving antibiotic treatment. RESULTS--The median stool outputs during the first 24 hours (275 ml/kg body weight) and till diarrhoea stopped (296 ml/kg body weight) were significantly higher in patients receiving 200 mg doxycycline as a single dose than in patients receiving either standard tetracycline (242 ml/kg body weight and 254 ml/kg body weight) or 300 mg doxycycline (226 ml/kg body weight and 255 ml/kg body weight). Similarly, median consumption of oral rehydration solution (18.45 l) was significantly higher in patients receiving 200 mg doxycycline than in patients receiving either 300 mg doxycycline (16.10 l) or standard tetracycline (14.80 l). Almost equal numbers of patients in each group required unscheduled intravenous acetate solution to correct dehydration during antibiotic treatment. Patients treated with doxycycline (low or high dose), however, had more prolonged excretion of bacteria. CONCLUSIONS--A single 300 mg dose of doxycycline is as effective as the standard multiple dose tetracycline treatment for cholera in terms of stool output, duration of diarrhoea, vomiting, and requirement for oral rehydration solution.     

Development of an accelerated method of determining the antibiotic sensitivity of Cl. perfringens type A]

An express method for determination of antibiotic sensitivity in the strains of Cl. perfringens of type A using Soviet dry nutrient media and antibiotics is proposed. The criteria for estimation of the level of the antibiotic sensitivity of the causative agent of gas gangrene in short periods on the basis of comparison of the data of the antibiotic agar diffusion procedure and the antibiotic MIC were worked out. Twelve antibiotics and 45 collection strains of Cl. perfringens of type A were used in the experiment. The antibiotic agar diffusion method with the use of the nutrient media, microbial load and cultivation conditions developed by the authors is recommended for tentative determination of the antibiotic sensitivity in Cl. perfringens of type A for 4 hours. The use of the agar diffusion method and determination of the antibiotic MIC provided complete estimation of tha antibiotic sensitivity of Cl. perfringens of type A within not more than 24 hours.     

Determination of the biological activity of antibiotics by using dry nutrient media

The possibility of using available dry nutrient media Nos. 5 to 12 in assays of antibiotic biological activity with the agar diffusions method was studied with respect to benzylpenicillin, gramicidin S, kanamycin sulfate, kanamycin B, oleandomycin, novobiocin, tetracycline and erythromycin. The dry media were used instead of the respective media prepared with meat hydrolyzate. Optimal conditions of the assays on such media were determined.     

Susceptibility of the Lyme disease spirochete to seven antimicrobial agents.

The antimicrobial susceptibility of five Lyme disease spirochete strains (two human and three tick isolates) was determined. A macrodilution broth technique was used to determine on three separate test occasions the minimal inhibitory concentrations (MICs) of seven antibiotics. The Lyme disease spirochete was most susceptible to erythromycin with a MIC of less than or equal to 0.06 micrograms/ml. The spirochete was also found to be susceptible to minocycline, ampicillin, doxycycline, and tetracycline-HCL with respective mean MICs of less than or equal to 0.13, less than or equal to 0.25, less than or equal to 0.63, and less than or equal to 0.79 micrograms/ml. The spirochete was moderately susceptible to penicillin G with a mean MIC of 0.93 micrograms/ml. All strains were resistant to rifampin at the highest concentration tested (16.0 micrograms/ml).