Production of hydrolases by lactic acid bacteria and bifidobacteria and their antibiotic resistance

It was demonstrated that bifidobacteria and lactic acid bacteria B. adolescentis and Lactobacillus sp. synthesized extracellular enzymes cleaving glycoside bonds in the molecules of dextran, pectic acid, and soluble starch. The maximal production of extracellular β-galactosidase by B. adolescentis 91-BIM and 94-BIM at a rate of 0.08 and 0.03 U/mg per h was observed during the exponential growth phase at 5 and 12 h of cultivation, respectively. The cultures of bifidobacteria retained 60–70% of β-galactosidase and α-amylase activities after six months of storage. The bifidobacterium strains studied were resistant to amphotericin and aminoglycosides (gentamicin, kanamycin, and netromycin). The lactam antibiotics (ampicillin, benzylpenicillin, bicillin 3, bicillin 5, and carbenicillin), the preparations inhibiting protein synthesis at the level of ribosomes (lincomycin), RNA polymerase inhibitors (rifampin), cephalosporin, and Maxipime inhibited the growth of bifidobacteria. Rifampin, erythromycin, amphotericin, Maxipime, Fortum, doxycycline, levomycetin, streptomycin, and the aminoglycosides netromycin, gentamicin, and kanamycin did not have an effect on the growth of Lactobacillus sp., whereas semisynthetic derivatives of penicillin, carbenicillin and ampicillin, inhibited its growth as well as Oxamp and lincomycin. The lactam antibiotics benzylpenicillin, bicillin 3, and bicillin 5 inhibited the growth of lactic acid bacilli by 30–90%.     

Sensitivity of clinical Proteus strains to antibiotics and their combinations]

In 1976 isolation of Proteus from wounds of patients with various purulent processes amounted to 14.5 per cent. Serotypes 0-10, 0-3 and H-3 predominated among the isolates. Sensitivity of 35 clinical strains of Proteus to 10 antibiotics, furagin and nevigramone was studied by the method of serial dilutions in liquid media. All the isolates were highly resistant to the antibiotics except gentamicin, furagin and nevigramone, the MIC of which for most of the strains was 3.12, 1.6-3.12 and 6.25-12.5 gamma/ml, respectively. The effect of 14 combinations of chemotherapeutics was also studied. The combinations of gentamicin with carbenicillin, gentamicin with ampicillin and monomycin with ampicillin proved to be most effective against the Proteus strains tested. The following combinations may be of practical value: monomycin + carbenicillin, kanamycin + ampicillin, kanamycin + carbenicillin, ampicillin + furagin, gentamicin + nevigramone. The combinations of carbenicillin with furagin and gentamicin with furagin were also rational.     

Interaction of aminoglycosides and the other antibiotic on selected bacterial strains

The aim of this study was to analyse the activity interaction of aminoglycosides (gentamicin, kanamycin, streptomycin and dihydrostreptomycin) when combined with other antibiotics (lincomycin, benzylpenicillin, amoxicillin, cephalexin, spectinomycin and erythromycin), on selected clinical bacterial strains. The checkerboard method has been selected from the traditional assays for the measurement of antibiotic interaction. Checkerboard results for all strains demonstrated synergism for nine cases (9/112--8%). Additive effects were predominant--73/112--65.2%. In 12.5% neutral effects were shown, but in 11.6% of combinations FIC indexes were not possible to calculate, because of the resistance of clinical strains to the highest concentration of at least one antibiotic. The best results were achieved for combinations of dihydrostreptomycin with procaine penicillin because of higher number of cases synergy effect was observed. Antagonism of aminoglycosides and beta-lactams in case of gentamicin and amoxicillin for E. coli and E. cloacea strains were shown. Potential activity for combination of streptomycin and erythromycin was shown.     

The sensitivity to 12 antibiotics of 125 strains of Streptococcus group B isolated in a maternity home

Sensitivity of 125 strains of group B streptococci isolated from newborns, their mothers and personnel in a maternity home was studied with respect to 12 antibiotics: benzylpenicillin, ampicillin, methicillin, cephalotin, erythromycin, lincomycin, levomycetin (chloramphenicol), oxacillin, tetracycline, streptomycin, gentamicin and ristomycin. The method of serial dilutions in a solid medium was applied. All the strains were sensitive to ristomycin and erythromycin. The predominating number of the strains were sensitive to lincomycin, levomycetin and the beta-lactam antibiotics. Strains resistant or moderately resistant to benzylpenicillin, ampicillin, oxacillin, methicillin and cephalotin were detected. The majority of the strains were resistant to streptomycin, tetracycline and gentamicin. Multiple antibiotic resistance with 2-7 determinants was revealed in 11.2 per cent of the strains. The antibiotic sensitivity of the strains isolated from the newborns, their mothers and the personnel in the maternity home was on the whole similar or insignificantly differed.     

Inactivation of aminoglycosides by beta-lactams of the penicillin and cephalosporin groups

Interaction of carbenicillin with kanamycin, gentamicin, tobramycin, and amikacin, as well as that of cefotaxime with the same aminoglycosides were studied. It was shown that carbenicillin inactivated aminoglycosides in a patient's plasma, urine, rH 5.3-8.44, and in buffer solutions, pH 3.7, 7.5 and 9.25. The inactivation level was the least in acid media. The mechanism of the inactivation was elucidated: opening of the carbenicillin beta-lactam ring occurred at the start.     

Study of the physiological and biochemical characteristics of bifidobacteria at the late stages of population development

An investigation of the physiological and biochemical characteristics of the Bifidobacterium bifidum no. 1, B. adolescentis MC-42, and B. adolescentis 94-BIM strains showed that bifidobacteria with a higher growth rate produced greater amounts of the end fermentation products, acetate and lactate. The growth of the strains in batch cultures was found to be inhibited by acidic fermentation products. The growth of B. bifidum no. 1 in a batch mode lasted 100 h at a population density of 10(6) CFU/ml and the growth of B. adolescentis MC-42 and 94-BIM lasted 96-120 h at population densities from 10(4) to 10(7) CFU/ml. Analysis of the bifidobacterial populations by light and electron microscopy showed that they represent conglomerates of cells with lysed cytoplasm in the cell center and intact cytoplasm in the apical parts of the cells. The maximum production of extra-cellular and cell-bound proteinases was observed in the logarithmic growth phase. By the 120th h of cultivation, the metabolic activity of cells, the production of proteinases, and the protein content of bifidobacterial cultures considerably decreased. In the first, second, and third subcultures of 96-h-old bifidobacterial cells on fresh nutrient media, the population density of bifidobacteria and their normal physiological and biochemical characteristics were restored after 48 to 72 h of cultivation.     

Sensitivity to antibacterial preparations of the pseudotuberculosis bacteria isolated on the territory of the Ukraine]

Sensitivity of 92 strains of the causative agent of pseudotuberculosis isolated in the Ukraine was studied with respect to 26 antibacterial drugs. It was found that the strains of the pseudotuberculous bacteria were sensitive to 17 drugs, i.e. benzylpenicillin, ampicillin, carbenicillin, cephaloridin, chloramphenicol, tetracycline, streptomycin, neomycin, monomycin, kanamycin, gentamicin, polymyxin, colistin, furadontin, nalidixic acid, sulfisoxazol and septrin. No differences in the sensitivity of the strains isolated in various districts of the Ukraine and from various sources were found. By their antibiotic sensitivity the strains isolated in the Ukraine did not differ from the cultures isolated in other districts of the USSR and abroad.     

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.     

Sensitivity of the wound microflora of oncological patients to some new antibiotics]

Sensitivity of the microflora of the oncological patients' wounds to the new antibiotics, such as gentamicin, kanamycin, oxacillin, ampicillin and lincomycin was studied with the help of the disc method. The discs with the above antibiotics were prepared under laboratory conditions in accordance with the respective instructions in the WHO. Sensitivity of 429 bacterial cultures, including 98 cultures of pathogenic staphylocci, 45 cultures of Enterococci, 43 hemolytic streptococci, 143 cultures of Escherichia, 50 cultures of Ps. aeruginosa and 50 cultures of Proteus was determined. The studies showed that gentamicin was the most active antibiotic aganist all the microbial species isolated from the surgical and other wounds of oncological patients. It may be used in treatment of the infections caused by association of the microbes belonging to different species, as well as in treatment of purulent processes before elucidating their etiology, 16.7 per cent of the Enterococcal isolates were resistant to gentamicin. Monomycin, kanamycin, oxacillin, lincomycin and macrolide antibiotics at present are sufficiently active against pathogenic staphylococci and hemolytic streptococci.     

Antibiotic sensitivity of nonfermenting gram-negative bacteria

Antibiotic resistance of 132 strains of nonfermenting gram-negative bacteria (NGNB) was studied. 43, 20, 17, 14 and 12 of them belonged to Acinetobacter calcoaceticus (anitratus and lwoffi), Pseudomonas cepacia, Alcaligenes faecalis, P. stutzeri and P. maltophilia, respectively. With rare exceptions all the strains were resistant to benzylpenicillin, oxacillin, lincomycin, ampicillin and cephaloridine. Sensitivity to the other antibiotics varied within wide ranges. Amikacin (94.3 per cent) and tobramycin (90.8 per cent), as well as polymyxin, rifampicin and gentamicin (71.7-66.9 per cent) had the highest effect. The majority of the antibiotics had higher activity (p less than 0.01) against the tested NGNB as compared to their activity against P. aeruginosa. Antibioticograms of every of the tested species of NGNB revealed that P. cepacia and P. stutzeri were the most resistant species. The biovars of Acinetobacter varied in their antibiotic resistance: A. subsp. lwoffi was more sensitive to the majority of the antibiotics though some of them, i.e. doxycycline, carbenicillin, and polymyxin were more active against A. subsp. anitratus.     

Inhibition of superoxide-dependent processes by aminoglycoside antibiotics

The ability of various antibiotics to inhibit superoxide anion(O-2)-mediated formation of adrenochrome from adrenaline and recovery of cytochrome c by xanthine oxidase was studied. In the adrenaline system (pH 10.2), aminoglycosides might be arranged, according to the inhibitory effect, in the following order: monomycin greater than gentamicin greater than kanamycin greater than lincomycin greater than streptomycin. In the xanthine oxidase system (pH 7.8), that order was the following: monomycin greater than gentamicin greater than lincomycin greater than greater than kanamycin. It was suggested that the antibiotic inhibition of the O-2-dependent processes at the essential sites of metabolism and/or the antibiotic involvement into the process of free radical oxidation initiated by O-2 in the cells might be one of the mechanisms of the drug action and toxicity with respect to the host.     

Comparative analysis of antibiotic sensitivity of Streptococcus pneumoniae strains isolated from patients and carriers

The data on antibiotic sensitivity of 38 strains of S. pneumoniae isolated from children and 46 strains isolated from carriers are presented. The isolates from the carriers had significantly higher sensitivity to benzylpenicillin, ampicillin, methicillin, oxacillin, cefazolin, erythromycin, oleandomycin and lincomycin. Resistance to gentamicin was more frequent in the strains isolated from the carriers. Among the strains of S. pneumoniae isolated from the patients and carriers representatives of serovar K19 were more frequent. There were no statistically reliable difference in them by sensitivity to benzylpenicillin, ampicillin, cefazolin, lincomycin and rifampicin. Still, the isolates from the carriers were much more sensitive to methicillin, oxacillin, oleandomycin and erythromycin.     

Sensitivity of Sherman's propionic acid bacilli to antibacterial preparations and vitamin B12 synthesis

Sherman propionic acid bacilli were sensitive to benzylpenicillin, ampicillin, ceporin, tetracyclines, oleandomycin, oletetrin, tetraolean, sigmamycin, levomycetin and furadonine. Methicillin, oxacillin, monomycin, kanamycin, polymyxin and furazolidone had an insignificant effect on the above organism. The subbacteriostatic concentrations of methicillin, oxacillin, streptomycin, monomycin, kanamycin, neomycin, tetraolean, sigmamycin, polymyxin M and ristomycin increased the biosynthesis of vitamin B12 by Sherman propionic acid bacilli, while benzylpenicillin, ampicillin, tetracyclines, oleandomycin, oletetrin, levomycetin and furadonine in the subbacteriostatic concentrations inhibited this process.     

Antibiotic sensitivity of the causative agents of suppurative surgical infection]

Sensitivity of 1492 strains of the causative agents of the surgical purulent infections, i. e. pathogenic Staphylococcus, Proteus, Ps. aeruginosa and E. coli to benzylpenicillin, streptomycin, levomycetin, tetracycline, erythromycin, oleandomycin, monomycin, kanamycin, novobiocin, ampicillin, oxacillin, ceporin, gentamicin and rifampicin was studied. Gentamicin was most active against all the bacterial species tested. The staphylococci were in addition sensitive in a high percentage of the cases to rifampicin, novobiocin, ceporin, monomycin and kanamycin. The isolates of E. coli were in addition sensitive to ceporin, monomycin and kanamycin. Sensitivity of the strains of Ps. aeruginosa and Proteus was low to all of the antibiotics except gentamycin. Most of the strains of the causative agents of the surgical purulent infections were multiresistant to 4 antibiotics. The number of the staphylococcal strains sensitive to benzylpenicillin, streptomycin and levomycetin increased in 1976 as compared to 1975 on the background of a limited use of these antibiotics in clinics.     

Patterns of growth and beta-galactosidase production by bifidobacteria

We investigated the patterns of growth and beta-galactosidase formation in the strains Bifidobacterium adolescentis GO-13, MS-42, 91-BIM, and 94-BIM, and B. bifidum No. 1, LVA-3, 791 on media with various carbon sources. The synthesis of beta-galactosidase was shown to be associated with exponential growth of the cultures involved. The maximum specific rate of beta-galactosidase synthesis of 0.20 U mg(-1) h(-1) was observed in B. bifidum LVA-3 after 3-6 h of cultivation. This value for B. adolescentis 91-BIM and 94-BIM was lower and amounted to 0.03-0.08 U mg(-1) h(-1). On the medium with lactose, the highest specific growth rates for B. bifidum LVA-3 and B. bifidum No.1 were 0.38 and 0.60 h(-1), respectively, after 3-6 h of cultivation. For B. adolescentis 91-BIM and 94-BIM, this parameter peaked at 12-15 h of cultivation at 0.13 and 0.22 h(-1), respectively. The hydrolytic activity of beta-galactosidase in the growth medium decreased during the stationary growth phase of the tested cultures.     

Sensitivity of Shigella to antibiotics]

Three hundred and twenty two Shigella cultures isolated from dysentery patients within 1986-1989 were tested with the use of standard paper disks for their sensitivity to levomycetin, streptomycin, tetracycline, monomycin, neomycin, kanamycin, erythromycin, gentamicin, carbenicillin, ampicillin, oxacillin, methicillin and doxycycline. The number of the cultures belonging to Shigella sonnei amounted to 85.1 per cent of the total number of the strains studied. 91.9, 89.5, 87.3, 87.3, 80.1 and 80.1 per cent of the cultures were sensitive to gentamicin, kanamycin, carbenicillin, neomycin, levomycetin and ampicillin, respectively. 99.4 per cent of the isolates were resistant to streptomycin and 97.2 per cent were resistant to tetracycline. The sensitivity to erythromycin remained rather high (70.2 per cent). The overwhelming majority of the Shigella sonnei isolates had multiple resistance.     

Antibiotic interaction with proteolytic enzymes]

The effect of penicillin, tetracycline, aminoglucozide antibiotics and streptomycin on BAEE-esterase activity of trypsin was studied. It was found that benzylpenicillin in amounts of 50, 100 and 300 mg, ampicillin in an amount of 25 mg, methicillin in an amount of 12 mg and tetracycline in an amount of 2.5 mg as calculated per 1 mg of trypsin had no effect in vitro on the esterase activity of the enzyme. Neomycin, kanamycin and streptomycin in amounts of 5, 10, 100 or 300 mg per 1 mg of trypsin catalyzed splitting of BAEE by trypsin. When the antibiotics were added to the bile, its esterase activity increased. Preliminary intramuscular administration of trypsin and kanamycin to the rats had no effect on the ampicillin levels in the blood serum and brain and did not affect the permeability of the hemato-encephalic barrier as compared to the use of trypsin alone.     

Method of oblique illumination in studying the morphology of Staphylococcus aureus colonies sensitive and resistant to antibiotics]

The morphology of the colonies of Staph. aureus sensitive and resistant to antibiotics and the colonies with artificial resistance to benzylpenicillin, ampicillin, oxacillin, methicillin, cephaloridin and lincomycin was studied with the method of slanting light. Changes in the morphology of the colonies were most pronounced at the beginning of the microbial resistance development first of all in the cultures of Staph. aureus resistant to cephaloridin and lincomycin.     

Comparative study of the antibacterial activity of aminoglycoside antibiotics and their combinations with carbenicillin against Pseudomonas aeruginosa

Antibacterial activity of 7 aminoglycoside antibiotics and combinations of tobramycin or gentamicin with carbenicillin was studied with respect to 33 clinical strains of Ps. aeruginosa. Tobramycin, sisomicin, gentamicin and amicacin showed high levels of antibacterial activity. Tobramycin and sisomicin were 3-4 and 2 times more effective than gentamicin. 100 per cent of the Ps. aeruginosa isolates was sensitive to tobramycin and amicacin. The number of the isolates sensitive to sisomicin and gentamicin amounted to 97 and 94 per cent respectively. The respective numbers for streptomycin and kanamycin were 32 and 11 per cent. No monomycin sensitive isolates were detected. Combination of tobramycin or gentamicin with carbenicillin increased the antibacterial activity of the aminoglycoside antibiotics by 2-16 times and that of carbenicillin by 2-32 times. The synergistic effect of gentamicin or tobramycin with carbenicilin was observed with respect to 50 and 58 per cent of the isolates respectively. No antagonistic effect was detected on the combined use of the antibiotics. The majority of the isolates (96 per cent) were sensitive to combinations of carbenicillin in a concentration of 50 micrograms/ml with tobramycin or gentamicin in concentrations of 0.15 or 0.3 micrograms/ml respectively.     

Purification and characterization of a beta-lactamase from Haemophilus ducreyi in Escherichia coli

A pCb plasmid encoding a beta-lactamase from Haemophilus ducreyi was transferred to Escherichia coli, purified, and characterized. The beta-lactamase could be isolated from a culture filtrate and further purified by ammonium sulfate and chelating Sepharose fast flow loaded with Zn(2+). The purified enzyme resulted in a major band at approximately 30-kDa on SDS-PAGE and its pI was determined to be 5.4. The beta-lactamase could hydrolyze both penicillin antibiotics including ampicillin, benzylpenicillin, and carbenicillin as well as cephalosporin antibiotics including nitrocefin, cephalothin, cephaloridine, and cefoperazone. However, benzylpenicillin was the best substrate. The enzyme activity was inhibited by clavulanic acid but not by boric acid, cefotaxime, ethylenediaminetetraacetic acid, or phenylmethylsulfonyl fluoride. The sequence of the beta-lactamase gene was also determined. It confirmed that the enzyme belonged to a class A beta-lactamase which had 99% identity to the ampicillin resistance transposon Tn3 of pBR322. Two nucleotides were different between the E. coli (Tn3) and H. ducreyi (pCb) genes that affected the amino-acid sequence. The valine at position 82 (ABL 84) was changed to isoleucine and the alanine at position 182 (ABL 184) was changed to valine. Genetic homogeneity among beta-lactamases is remarkable. Amino acid sequencing of some beta-lactamases has shown that substitution of only a few amino acids in the bla gene leads to high-level resistance against specific cephalosporins.