Search

Investigation of the properties of antibiotic-resistant variants obtained experimentally from various species of Brucella indicated that benzylpenicillin, ampicillin and ceporin induced significant changes in the main properties of Brucella, i.e. phagolysability, agglutinability, production of brucellacin and hydrogen sulfate, attitude to dyes. Only the nutrient requirements of Brucella did not change when it was grown on synthetic media which is of great importance for investigation of changed Brucella strains.

The kinetics of inhibition of penicillinase of Bac. licheniformis 749/c by various alkyl-sulphates (with a long hydrocarbon chain from C8 to C16), n-toluolsulphoacid and alkylbenzol-sulphonate (R-C12--C16) was studied. The inhibition rate increased with elongation of the alkyl radical and rising of the inhibitor concentraiton. This means that the determining factor in inhibition process was hydrophobic interaction of the alkyl chains and not the electrostatic interaction with the enzyme. In-vitro experiments with penicillinase-producing strains of staphylococcus showed that non-bactericidal concentrations of the alkylsulphates and alkylben zolsulphonates increased the effect of benzylpenicillin, ampicillin and methicillin. The highest increase in the antibiotic activity, as well as in the enzyme inhibition was observed with respect to the compound with the hydrocarbon chain fron C12 to C16. The increase in the activity of methicillin against the staphylococcal strains resistant simultaneously to benzylpenicillin, ampicillin and methicillin was indicative of possible using of the above surface active substances as inhibitors of the realization process of various mechanisms of penicillin resistance.

Transduction of R-factors with phage Rlkc was studied. Six of the R-factors controlled resistance to single antibacterial drugs, such as tetracycline, levomycetin, ampicillin, neomycin, streptomycin and nitrofurans. When the donor strains produced bacteriocins, treatment of the transducing lysates with trypsine increased the yield of the R+-transductants. Transduction of the resistance determinants was not in most cases accompanied with lysogenization of the recipient cells. It was confirmed that transduction with phage Rlkc provided division of the R-factors into 2 groups, i. e. those with the resistance determinants possessing transferability after transduction and those with the determinants possessing no such ability. It was supposed that one of the causes of the R+-transductant inability to transferance of the resistance determinants on conjugation was their intergration into the recipient chromosomes on transduction.

It was found that clinical strains of Ps. aeruginosa produced inducable beta-lactamase in the presence of penicillins, such as benzylpenicillin, ampicillin, carbenicillin. The amount of beta-lactamase depended on the strain and the inductor type and increased with the inductor concentration increase. Benzylpenicillin was the most active inductor of the enzyme synthesis by Ps. aeruginosa, strain 649. In concentrations of 5000 gamma/ml it provided at least 100 times higher amounts of beta-lactamase as compared to the initial level. The dynamics of induction of the enzyme synthesis by Ps. aeruginosa, strain 649 on addition of benzylpenicillin (5000 gamma/ml of the medium) at various stages of the culture growth was studied. Maximum accumulation of induced beta-lactamase was observed on addition of benzylpenicillin to the medium 2 hours after the culture inoculation. The induced enzyme was mainly found in the bacterialcells. The culture treatment with toluol increased the amounts of the determinable induced enzyme 4-26 times, 30 to 80 percent of the induced enzyme were liberated from the cells into the supernatant. Dependence of the level of the culture resistance to benzylpenicillin and ampicillin on the induction rate of beta-lactamase synthesis provided by the penicillins was found. No such dependence was observed with respect to the culture resistance to carbenicillin.

The study of the staphylococcal population heterogeneity with respect to methicillin resistance by 2 methods revealed different numbers of the resistant cells in the population. Thus, when the microbial suspension was plated on an agarized medium with methicillin (50 gamma/ml), only 0.0007--0.0005 per cent of the resistant cells were found. When the colonies were replicated from a medium without methicillin to a medium containing methicillin (50 gamma/ml), 84.3--97.3 per cent of the resistant microbial cells were found in the population of the same strains. The main mechanism in the heterogeneity of the staphylococcal population with respect to methicillin resistance was impairement of the phenotype manifestation of the antibiotic resistance under definite conditions.

The literature data and personal observations of the authors on the effect of the inoculate amount on the results of determination of microbial sensitivity to antibiotics by the methods of serial dilutions in the liquid and solid nutrient media and agar diffusion are discussed. It was shown that the inoculate of the density of 3.6.10(7) to 4.25.10(7) microbial bodies per 1 ml was optimal for the methods of agar diffusion and serial dilutions in agar. Recommendations for simplifying standardization and dilution of the inoculate are presented.

The dynamics of staphylococci sensitivity to antibiotics in purulent foci of 242 in patients was studied. Two criteria were used for estimation of the above process, i.e. (I) sensitivity to the antibiotics of the whole staphylococcal population isolated and (2) sensitivity to the antibiotics of separate strains isolated from the population. In 10 or more days the number of the in-patients with staphylococci resistant to streptomycin, monomycin or oxacillin in the purulent foci increased. The number of the in-patients with penicillin, neomycin or chloramphenicol resistant staphylocci was statistically doubtful. The average number of the antibiotic resistant microbial cells in the staphylococcal populations of the foci in the patients during their stay in the hospital increased 2-4 times.

The antimicrobial effect of benzylpenicillin in combination with bile acids on staphylococci was studied. It was shown that bile acids increased the bacteriostatic and bactericidal effects of the antibiotic. The highest potentiating effect was found with combination of benzylpenicillin and desoxycholic and choleic acids. When resistant strains of staphylococci were exposed to the combination of the bile acids and benzylpenicillin they became highly sensitive to antibiotic.

Interaction of benzylpenicillin inactivation by beta-lactamase and the growth of E. coli in the presence of various antibiotic concentrations was studied. It was found that penicillin inactivation began immediately after addition of the culture to the medium in spite of the fact that the number of the viable cells at that time decreased. The culture began to multiply only when benzylpenicillin concentration in the medium lowered up to a definite level (6-7 gamma/ml). Such a concentration must determine the level of the "inner" culture resistance to benzylpenicillin, not connected with the beta-lactamase effect. Moreover, the "inner" resistance proved to be the same for the cultures of E. coli, markedly differing by the amounts of beta-lactamase produced and the resistance levels to benzylpenicillin. Therefore, the routine method for determination of the "inner" resistance of the beta-lactamase-producing cultures to penicillins, using low innoculation doses produced higher results as compared to the real "inner" resistance. Significant differences observed between the values of the "inner" resistance (6-7 gamma/ml) and the MIC of benzylpenicillin at high innoculation doses with respect to E. coli 2 (129 gamma/ml) and E. coli 82 (15000 gamma/ml) may probably be explained by destruction of the drug by beta-lactamase.

Incorporation of transposone Tnl of ampicillin resistance into the chromosome of E. coli C600 resulted in formation of at least 8 types of auxotrophic mutants. The transposone incorporates mainly into the loci the damage of which induces proline deficit (20 per cent). The sites of incorporation of transposone Tnl in the chromosomes of E. coli C600 and E. coli JC411 did not coincide.

The experiments showed that among 110 Salmonella strains resistant to 1 or more antibiotics 54.5 per cent of the cultures transfered their resistance to E. coli when grown in vitro. The frequency of the transmissive drug resistance in Salmonella was directly proportional to the number of r-determinants in R-factor. The cultures resistant to ampicillin and kanamycin proved to be the most competent donors.

Occurrence of the markers of penicillin and erythromycin resistance in the clinical strains of Staph. aureus, as well as their connection with the determinants of resistance to the heavy metal salts and sodium arsenate was studied. 79 per cent of Staph. aureus were resistant to penicillin and 47 per cent to erythromycin. All erythromycin resistant strains were also resistant to penicillin. More than a half of the strains of Staph. aureus of the phage groups I, III and the mixed phage group had a set of markers: pen, asa, cad, mer, ego. The rate of elimination of the above markers with ethidium bromide was high. The presence of the penicillinase plasmid determining a rather low level of resistance to erythromycin (20--50 gamma/ml) was characteristic of the strains belonging to the phage group I. The presence of the plasmid determining a high level of resistance to that drug (500--1000 gamma/ml) was characteristic of the phage group III and the mixed phage group. The erythromycin sensitive strains of Staph. aureus were almost always sensitive to mercuric ions.

Resistance parameters of 17 clinical strains of Staph, aureus to oxacillin were studied with the methods of agar diffusion and serial dilutions in liquid and solid media. Statistically reliable correlation between the parameter values of resistance and growth inhibition zone diameters on diffusion into agar of 100 gamma of oxacillin was found. The latter method provided classification of the strains into groups with high, intermediate and low resistance levels. However, calculation of the resistance parameters according to the corresponding correlation equation was not enough correct for providing distinction in the resistance levels within the groups.

The structure of drug resistance was studied on 2788 clinical strains of staphylococci isolated from various materials of patients with purulent-inflammatory processes, stuff and environment within 12 years from 1968 to 1973. The number of staphylococci resistant to tetracycline increased 13 times among the cultures with resistance marker, while the number of staphylococci resistant to penicillin decreased 1-8 times. The study of the complex resistance revealed the predominance of staphylococci with tetracycline resistance structure (60-5 percent).

Penicillin sensitivity of gonococci isolated from patients with relapses and reinfection of gonorrhea was studied. The results of the study were compared with those of the sensitivity tests in primary patients. The gonococcal strains with decreased sensitivity to penicillin were isolated from 82 per cent of the gonorrhea relapses, 47.8 per cent of the patients with reinfection and 33.2 per cent of the primary patients. The average sensitivity of the gonococcal strains isolated from the patients with relapses was 0.404 U/ml, while that in the primary patients and the patients with reinfection was 0.136 U/ml. Strains sensitive to 0.6--1.2 U/ml predominated among gonococci with decreased sensitivity in the patients with relapses, while in the primary patients and the ones with reinfection the value amounted to 0.1--0.2 U/ml. The penicillin sensitivity of gonococci may be used as a parameter in differential diagnosis of relapses and reinfection of gonorrhea.

Development of resistance to penicillins and streptomycin in bacteria results in an increase in the electrokinetic potential changes in the hydratation rate and isoelectric levels of pH. Changes in the physico-chemical properties of the surface of penicillin-resistant Escherichia depends mainly on the accompanying dissociation. The ability of Escherichia to bind labeled benzylpenicillin is shown. Binding of 35S-benzylpenicillin by Escherichia in S-form correlates with sensitivity of the culture to the antibiotic. The penicillin-resistant mutants in R-form bound the antibiotic to a greater extent than sensitive R-cultures. The role of penicillinase in binding 35S-benzylpenicillin by Escherichia was not shown experimentally. Only increased synthesis of the enzyme by the penicillin-resistant mutants was observed. The ability of Escherichia to bind 35S-benzylpenicillin does not depend on the physico-chemical properties of the cell surface and dissociation.

Chemotherapeutic activity of various benzylpenicillin combinations with 10-nitro-so-trans-anti-cys-perhydroacridine (MT-2) was studied on 2 experimental staphylococcal infections caused by pathogenic penicillin resistant strains of staphylococci. It was found that the combined use of the antibiotic and MT-2 in doses of 25-100 and 12.5 mg/kg respectively administered subcutaneously increased the therapeutic effect of benzylpenicillin as compared to the antibiotic used alone. The oral use of MT-2 in a dose of 400 mg/kg also increased the therapeutic action of benzylpenicillin used in a dose of 100 mg/kg subcutaneously simultaneously with MT-2. A favourable effect of ointments containing MT-2 in a concentration of 10-20 per cent and benzylpenicillin in a dose of 50 000 or 100 000 units per 1 g of the ointment was noted in treatment of mice with localized staphylococcal infection.

The in vitro study of the possible formation of L-forms of Brucella under the effect of tetracycline, streptomycin, rifampicin or their combinations with penicillin showed that passages of various Brucella species on media containing increasing concentrations of the antibiotics resulted in insignificant morphological changes in the cells. Passages of the same cultures on media containing combinations of the above antibiotics with penicillin resulted in more significant changes in the cell morphology, up to formation of spheroplasts. No L-transformation was observed under the experimental conditions.

The dynamics of sensitivity to penicillin of staphylococcal populations in purulent inflammatory foci of patients treated and not treated with antibiotics was estimated according to 4 indices. No reliable differences in the dynamics of sensitivity to penicillin were found in 2 groups of the patients, when estimation was performed with respect to the frequency of the penicillin resistant or penicillin sensitive staphylococci and detection of the penicillin resistant staphylococci by direct inoculation of the focal excretion to the medium with penicillin. A reliable increase in the percentage of the penicillin resistant staphylococci in the microbial population was observed only in the patients treated with penicillin.

Transmissivity of drug resistance in wild strains of E. coli was shown to be determined not only by the presence of the transmissive factor and r-determinants in the microbial cell, but also by the functional activity of the R-factor. The capacity of the R-factor for transmission depended to a certain extent on the number of the r-determinants in the R-factor and the presence of various r-determinants in the R-factor.

beta-Lactamase and penicillinacylase were detected in the tularemia causative agent. These enzymes participated in the mechanism of natural resistance of the microorganisms to penicillins. It was shown that the growth temperature had a significant effect on the beta-lactamase activity. The penicillin resistance marker was not eliminated with the use of acridine orange, ethidium bromide or sodium dodecylsulfate.

Morphological changes in phagocytized cells of Staph. aureus due to methicillin were studied with electron microscopy of ultrathin sections. Peritoneal macrophages of mice obtained by excitation of the abdominal cavity with medium No. 199 followed by infection with 1-milliard staphylococcal suspension were tested. Methicillin was administered in single doses of 2.5, 5, 25 and 50 mg/kg. The doses of 2.5 and 5 mg/kg induced specific changes in some cells. First of all the division septa were affected. After that the cell walls became thinner the cells increased in size, the cytoplasm became electronically less solid. No lysed cells were detected. The use of the doses of 25 and 50 mg/kg entailed polymorphism of the damages. Perforations formed in the bacterial cell wall. The cytoplasm acquired a characteristic granular structure evident of the cell death. With an increase in the dose from 25 to 50 mg/kg no rise in the level of the morphological changes in the bacterial cells was noted. Determination of methicillin concentration in the peritoneal exudate showed that it was 2--4 times higher depending on the dose than the MIC in vitro.

Biochemical activity of the guinea pig leucocytes phagocyting live antibiotic sensitive and resistant bacteria was studied in vitro. The results of the study showed that the leucocytes which had absorbed live Staph. aureus and E. coli sensitive and resistant to be benzylpenicillin and levomycetin respectively differed in increased contents of nucleic acids. The increase was due to higher contents of RNA. The activity of dehydrogenases, acid and alkaline phosphatases was markedly inhibited. The use of oxygen and lipase activity did not differ from the control in the experiments with phagocytosis of both Staph. aureus and E. coli. The activity of peroxidase and catalase in the experiments with phagocytosis of E. coli did not differ from the control, while in the experiments with phagocytosis of Staph. aureus it increased. The data testified to the fact that the enzymatic activity of the phagocytes changed when they absorbed the objects of phagocytosis, the changes in the biochemical activity being dependent on the nature of the antigen and its state.

Integration of R-factor RP4 with the chromosome of E. coli was studied with the use of replication thermosensitive mutant pEG1 of this factor. It was found that the frequency of integration of factor pEG1 containing the ampicillin transposone Tn1 with the chromosome of bacteria JC411 carrying transposone Tn1 previously inserted into it was very high and markedly exceeded that of its insertion into the same chromosome but not carrying this transposone. The frequency of factor pEG1 insertion into the chromosome of bacteria JC 1553 rec A defective with respect to genetic recombination was less than 2.10(-5) and did not depend on the presence of transposone Tn1 in it. Probably, insertion of factor RP4 into the bacterial chromosome may be realized through the rec A-dependent process of recombination between transposone Tn1 previously translocated into the chromosome and the same transposone contained in R-factor.

Bacteriological analysis of urine of 150 patients with chronic pyelonephritis was performed. As a result mycoplasma was isolated from urine of 25 patients. Mycoplasma and Coli bacteria or Proteus were isolated simultaneously from urine of 10 patients. Biochemical properties and sensitivity to antibiotics of 9 isolates were studied. The data provided recommendation of the urine analysis for the presence of mycoplasma.

Sensitivity of 33 foreign and 10 native strains of Y. enterocolitica to tetracycline, aminoglycosides, penicillins, levomycetin and polymyxin was studied. All the strains proved to be resistant to penicillin, oxacillin and ampicillin: they produced penicillinase. The level of resistance to penicillin did not always correlate with penicillin activity. The ability of the native strains to acquire R-factor in vitro from Coli bacteria was shown.

Sensitivity of 4 clinical strains of Staph. aureus and E. coli to 13 hydroacridine derivatives and their combinations with antibiotics, such as benzylpenicillin, ampicillin, semi-synthetic penicillins, streptomycin, chloramphenicol, tetracycline, chlortetracycline, monomycin, oleandomycin and erythromycin was studied. The highest bacteriostatic effect was observed on the use of perhydroactidine derivatives with benzylpenicillin or ampicillin with respect to polyresistant penicillinase-producing strains of Staph. aureus, resistance of which to these antibiotics was decreased 250--1000 times. Under the effect of the above compounds the staphylococcal resistance to chloramphenicol, tetracycline, chlortetracycline, oleandomycine and erythromycin decreased 2--66 times. The combinations of hydroacridine with the antibiotics, except 10-amino-trans-syn-trans-perhydroacridine had no effect on the resistance of the E. coli strains. The results of the combined effect of the above substances were associated with their chemical nature, the bacterial type and possibly the character of the strain resistance.

A total of 123 cultures of the genus Micromonospora and 89 cultures of the genus Streptoverticillium were studied for their capacity to produce beta-lactamase inhibitors. It was shown that streptoverticilla were highly resistant to beta-lactam antibiotics. No organisms producing beta-lactamase inhibitors were detected among the cultures of this genus. As for Micromonospora, 30 cultures (24.4 per cent) of this genus produced inhibitors. They were most frequently isolated from orange and brown cultures of Micromonospora. The inhibitors had no antibacterial activity, inhibited beta-lactamases of gram-negative bacteria and did not inhibit exogenic beta-lactamases of staphylococci. Their inhibitory activity was evident only with the use of isolated enzymes. With the use of intact growing cultures of gram-negative bacteria producing the same enzymes (Pseudomonas, Klebsiella, Enterobacter) no inhibitory activity was observed. Though the inhibitors had some features in common, they did not seem to be similar, since they had different spectra with respect to their effect on various beta-lactamases of gram-negative bacteria.