Search

The study was concerned with investigation of the biochemical properties, antibiotic resistance and the effect of various doses of penicillin on the activity of metabolic enzymes in the bacterial cells of Staphylococcus epidermidis isolated from the pharynx mucosa of healthy subjects. The study revealed an increase in the aggressive properties of the representatives of the mouth normal microflora. It was also observed that the highest percentage of the staphylococcal isolates was resistant to beta-lactam antibiotics. The effect of various concentrations of penicillin on the microbial metabolism was shown to be directed at different targets. Penicillin intensified the aerobic processes and lipid synthesis in the bacterial cells. The reactions of the processes of the amino acid metabolism proceeded in different directions. Some concentrations of penicillin inhibited such processes.

The microbiological surveillance of pneumococci according to the Protocols for the International Surveillance of Pneumococci Resistant to Penicillin and Other Antimicrobial Agents developed by Prof. M. Jacobs (USA) and Prof. P. Appelbaum (USA) made it possible for the first time in Russia to detect the strains with moderate resistance to penicillin. It is necessary to revise the current Russian recommendations for the laboratory diagnosis of pneumococcal infection and the determination of antibiotic susceptibility so as to participate in the united programme of the International Surveillance of Penicillin Resistance in Streptococcus pneumoniae.

Optimal conditions were developed for determination of antibiotic sensitivity in Brucella by using enzyme immunoassay directly in the primary cultures of the material tested. The Brucella concentration in the material tested should be not lower than 1.10(6) microbial cells/ml and the time of culture incubation be 24 hours at 37 degrees C. The obligatory condition is to use a liquid medium, i.e. the Albimi broth with 1% glucose. To inhibit the foreign microflora it is recommended to use polymyxin B and amphoglucamine in a concentration of 3 microgram/ml. The use of enzyme immunoassay was shown that it was possible to determine the antibiotic sensitivity of Brucella in practice.

The plasmid analysis of a collection of the staphylococci isolated was made in the obstetric hospitals of Nizhny Novgorod and Arzamas in 1986-1989. It revealed the presence of large complex plasmids with a molecular weight of 22 MD carrying the determinants of resistance to penicillins and cadmium and mercury ions in polyresistant strains. Conjugation transfer of the plasmid even between the staphylococci of different species was performed under experimental conditions. It was suggested that occurrence of the strains carrying the complex cadmium-penicillinase plasmid was due to violation of the sanitary or ecological requirements.

The resistance of methicillin-resistant staphylococci to phage 85 is due to the presence of a certain system restriction modification in microbial cells. The loss of the capacity for restricting phage DNA by the cell as the consequence of the loss of the mec determinant is not accompanied by the loss of its capacity for modifying phage DNA.

Altered viable forms of F. tularensis with spheroplast specific damages of the surface structures were isolated after the culture exposure to lithium chloride (0.5 and 1%). Study of natural penicillin resistance in the spheroplasts and bacterial forms of F. tularensis revealed their difference: the spheroplasts of the strains tested had a lower resistance to beta-lactam antibiotics than the bacterial forms while the activity of spheroplast beta-lactamase did not differ from that of the enzyme of the bacterial form and equalled 224 to 252 U/ml of the cell suspension. Therefore, on the model of the lithium-induced spheroplasts it appeared possible to show that the damages of the surface structures of the cell walls of F. tularensis changed the penicillin resistance level which was indicative of involvement of the F. tularensis cell walls in the phenomenon of the natural resistance to beta-lactams.

Sensitivity of different species of Lactobacillus i.e. L. casei, L. plantarum, L. acidophillus, L. buchneri, L. jugurti and others to penicillins and cephalosporins of various generations was studied. Penicillin binding proteins (PBPs) of the Lactobacillus species were specified. It was shown that the number of PBPs depended on the Lactobacillus species. L. casei had the least number of PBPs (4) and L. brevis had the highest number of PBPs (11). Competition of 14C-benzylpenicillin with ampicillin, cefotaxime, ceftizoxime and cefoperazone for binding to separate PBPs in three strains of different Lactobacillus species was investigated.

Sensitivity of 788 strains of Erwinia isolated from natural substrates and received from other laboratories was studied with respect to penicillin, streptomycin, tetracycline, chloramphenicol and kanamycin. It was found that 346 (43.9 per cent) strains were drug resistant. However, the levels of the resistance to most of the drugs tested were not high and amounted to 5-10 gamma/ml. Penicillin resistance was the most frequent characteristics of the strains studied (341 strains). The resistance levels to this drug were higher than those to the other drugs and ranged from 25 to 100 gamma/ml. In some cases they were higher. Analysis of the drug resistance spectra showed that Erwinia strains resistant to 1 (172 strains) or 2 (149 strains) antibiotics were most frequent. 25 strains were multiresistant (to 3-5 drugs). All the resistant strains had 18 types of drug resistance. The genetic control of the drug resistance feature was carried out in the experiments on crossing of the drug resistant strains of Erwinia with the sensitive bacteria of the same genera, i.e. Erwinia chrysanthemi EP-3 and E. coli K-12. The results of the experiments were negative, which should be considered as an evidence of chromosomal localization of the antibiotic resistant genes in the Erwinia bacteria studied.

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.

The studies on the effect of a number of antibiotics on transfer of R-factors with the use of a kinetic conjugation system showed that rifampicin inhibited the transfer of various R-factors independent of the microbial host strain and R-factor type (the inhibition coefficient of I infinity equals 7.5-16.8). The early conjugation stages preceding the transfer of R-factor DNA were most sensitive to the antibiotic effect. Decreased number of R+ conjugants observed on the donor strain growth in the presence of rifampicin were due rather to selection of spontaneous R- variants with the antibiotic found in our experiments than to its direct effect on the donor competence of R+ cells. It was also found that exposure of E. coli strains to rifampicin for 30 minutes increased their sensitivity to chloramphenicol and tetracycline, this being more pronounced in the cells which not long ago acquired the R-factor. The mechanism of the phenomenon is not clear yet. It was shown to be not associated with the cell permeability impairement under the effect of rifampicin. It was noted that rifampicin combination with neomycin chloramphenicol, tetracycline or streptomycin had a synergistic effect on the strains of E. coli.

A study was made of 111 strains of plasma-negative spathylococci isolated from the blood, pleural fluid, urine, and exudate of the abdominal cavity of 30 patients. The studies were carried out by 18 criteria. A variety of biological properties and signs characteristic of pathogenic staphylococci (hemolytic activity, anaerobic splitting of mannite, the presence of phosphatase, lysozyme, protease, alpha-toxin, fibrinolysin) were noted. A high resistance to tetracycline and penicillin was found in the strains isolated from the blood and the pleural cavity.

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.

beta-Lactamases of Proteus and their role in the mechanism of the microbe resistance to penicillins and ceporin were studied. It was found that the beta-lactamase of Proteus had low activity and were produced by both beta-lactamide resistant and sensitive clinical strains of Proteus. The resistant cultures of Proteus produced enzymes more frequently (3.4--5 times) than the sensitive ones. The synthesis of beta-lactamase in the clinical Proteus strains was inducable. The high induction coefficient was achieved only in the presence of high concentrations of the inductor. No significant dependence of the culture sensitivity level of ampicillin and ceporin on the induction level was observed. The most significant part of the constitutive enzyme in Proteus was intracellular, while that of the inducable enzyme was extracellular. No correlative dependence between the culture resistance levels to penicillins and ceporin and the enzyme activity was noted. The beta-lactamase activity was not found in the transconjugants with the in vitro acquired R-factor controlling the ampicillin and ceporin resistance, as well as in the resistant mutants selected on the media with increasing concentrations of the above antibiotics. Induction of beta-lactamase synthesis was not found in these strains either. The ability of Proteus to synthesize beta-lactamase can be lost on the strain storage under laboratory conditions which was not always accompanied by reduction of the culture sensitivity to ampicillin and ceporin. The enzymatic destruction of beta-lactamides was not the main mechanism of Proteus resistance to the above antibiotics.

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.

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.

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.

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).

S. derby K 89 isolated from sick children have been found to contain plasmids which determine the resistance of these organisms to penicillin and chloramphenicol. 3 types of plasmid DNA with molecular weights of 40. 12 and 8 megadaltons (Md) have been detected by electrophoretic analysis. The dissociation of the large plasmid (40 Md) into 3 independent replicons has been observed. The circular, covalently closed forms of the DNA cointegrate and its constituent replicons have been identified by electron microscopy. In S. derby K 89 grown in broth prepared from dried meat concentrate the loss of these plasmids is observed, and the culture thus becomes sensitive to penicillin and chloramphenicol. This suggests that dried meat concentrate contains some factor capable of eliminating the plasmids.

The role of penicillinacylase in resistance to penicillin of various strains of E. coli differing in the levels of acylase produced was investigated. All the strains produced simultaneously acylase and beta-lactamase. A preparation of pure acylase and an antiacylase immune serum were used in the experiments. The sensitivity testing was accompanied by chromatography of the culture broth with a purpose of finding penicillin inactivation products. A principle possibility of increasing the culture resistance to penicillin under the action of acylase was found. However, in testing the strain sensitivity with a method of serial dilutions the conditions for synthesis and action of acylase were unfavorable. Therefore, the main factor of the culture resistance to penicillin was beta-lactamase and not acylase.

Differences in the protein-composition of methicillin resistant (MR) and methicillin sensitive (MS) staphylococci were shown by means of disk electrophoresis. Extracellular proteins of MR and MS staphylococci were studied electrophoretically. The study revealed no relation between the culture grouping by the protein spectra and resistance to methicillin. Comparison of the spectra of soluble intracellular proteins showed a certain correlation with methicillin resistance.

The composition of fatty acids of methicillin-resistant (MR) and methicillin-sensitive (MS) strains of Staph. aureus and Staph. epidermidis was determined with the method of reactive gas liquid chromatography. The MS staphylococci of the above species differed by the content of acids with branched chains of iso- and anteisostructures and straight chains. Anteisoacids in the cells of Staph. epidermidis amounted almost to 80 per cent of the total number of the acids, while in the cells of Staph. aureus, their total number amounted only to a half of the fatty acid pool. Comparison of the composition of the fatty acids of the MS and MR strains of Staph. aureus revealed differences in the proportions of the anteiso- and isoacids. The total number of the long-chain C20.0 acid in the cells of Staph. epidermidis resistant to methicillin was lower as compared to that in the sensitive cells.

The chemotherapy of cholecystocholangitis in patients with virus hepatitis was estimated. Antibiotics, furagin and pathogenetic drugs were used for the treatment. The antibiotics were chosen with regard to their sensitivity to the bile microflora. This resulted in sanation of the bile ducts. Furagin was less effective. The use of the pathogenetic drugs alone was ineffective in the majority of patients.

Acute dysentery and other acute intestinal diseases with obscure etiology are accompanied by pronounced intestinal dysbacteriosis which is manifested, on one hand, by the increased levels of Escherichia, enterobacteria, staphylococci and by the appearance of Klebsiella and Proteus in large amounts and, on the other hand, by an essential decrease in the number of lactic acid streptococci, lactobacteria, bifidobacteria. The treatment of dysentery and other intestinal diseases with obscure etiology with antibiotics, while eliminating the infective agent, leads to the aggravation of intestinal dysbacteriosis. The combined use of antibiotics and antibiotic-resistant bifidobacteria in intestinal infections with obscure etiology contributes to the effective restoration of normal intestinal microflora already in the process of antibiotic therapy.

The effect of the incubation temperature, i.e. 37 and 30 degrees and the effect of addition of 5 per cent of sodium chloride to the nutrient medium was studied with respect to the results of sensitivity determination of staphylococci to methicillin by 3 methods, such as serial dilutions in liquid and solid media and replica method applied to separate colonies. With the use of all 3 methods incubation of the samples at a temperature of 30 degrees increased the rate of staphylococcal cultures resistant to methicillin. Addition of sodium chloride to the solid nutrient medium increased the level of detecting methicillin resistant cultures in the samples incubated at a temperature of 37 degrees and decreased it in the samples incubated at 30 degrees.

The study of 50 clinical strains of Ps. aeruginosa revealed their high resistance to benzylpenicillin, ampicillin, oxacillin, streptomycin and kanamycin with a tendency to polyresistance. The same strains were highly sensitive to gentamycin and polymyxin. The strains produced constitutive beta-lactamase in small amounts. The enzyme synthesis in Ps. aeruginosa was induced by benzylpenicillin. Still high concentrations of it were required for the induction. The maximum induction of beta-lactamase synthesis was observed by the 6th hour of the inductor addition. The maximum induction level was observed 4 hours after addition of benzylpenicillin. The level of induction of beta-lactamase synthesis in the strains ranged within 5-93. A significant part of the enzyme was liberated from the cells during induction. Interaction between the induction level of beta-lactamase synthesis and resistance of Ps. aeruginosa to penicillins was found.