Subinhibitory concentrations of organic ammonium salts: The effect on biological properties ofSalmonella typhimurium

The effect of subinhibitory concentrations (subMICs) of new organic ammonium salts of four homologous series of alkylammonium bromides (32 compounds) was determined with respect to the induction of lysogenic strain prophage, influence of permeability reactions in a rabbit skin test and cytotoxic changes of monolayers of Vero cells. The culture filtrates were prepared by 1-d cultivation ofSalmonella typhimurium in a synthetic culture medium under conditions of intensive aeration at 37°C after addition of subinhibitory concentrations of organic ammonium salts. The results showed that substances of the homologous series of 2-(10-undecenoyloxy)ethyl-alkyldimethylammonium bromides were characterized by a prophage-inducing effect in lysogenic strain cells. The induction of prophage raised with rising concentrations of subMICs of the substances, and its titer in the culture filtrates was mostly 4.10⁶ PFU/mL. SubstancesC3, C9 andC12 of the same homologous series had the strongest effect on the permeability reaction in rabbit skin in 1/2 MICs. One-half MICs of four substances (B14, C3, C12, C14) and 1/4 MICs of substanceA16 influenced cytotoxic changes on Vero cells, the other substances were ineffective.     

Effects of subinhibitory concentrations of nitroxoline on the surface properties of Escherichia coli

Nitroxoline (5-nitro-8-quinolinol; NIQ) at subinhibitory concentrations (sub-MIC) decreased the adherence of uropathogenicEscherichia coli to catheter surface and significantly enhanced cell surface hydrophobicity. The surface hydrophobicity increased in the presence of sub-MIC of NIQ and also in an excess of Mg²⁺. The effect of NIQ on the cell surface was not related to the bacteriostatic effect of this agent. The increase in nitrogen and decrease in phosphate content in the cell surface was found in the presence of NIQ. NIQ did not inhibit the expression of fimbriae.     

The effect of subinhibitory concentrations of antibiotics on the adhesion of enterobacteria

It is impossible to determine rigidly a net result of the influence of antibiotics on the interaction between parasite and host cells, as many factors participating in this process are not studied. Adhesion of microorganisms is one of the essential mechanisms of the above interaction. Antibiotics with a different mechanism of action in the subinhibitory concentrations affecting viability of microbes either slightly or nowise have been studied for their effect on adhesion on a model of the intestine section of human embryos and experimental animals. Most of antibiotics influenced differently adhesion of the microorganisms, that also depended on the species attribution of the latter. The accelerated selection of resistance during a successive passage via the suggested adhesion system was observed. The data obtained elucidated certain mechanisms of the effect of antibiotics on the microbial populations at the initial phase of the infectious process and under the primary contamination of mucosa.     

Inhibition ofPseudomonas aeruginosa alginate expression by subinhibitory concentrations of antibiotics

The effects of subinhibitory concentrations (1/4, 1/8, 1/16 of the MIC) of quinolones (ciprofloxacin, enoxacin, nalidixic acid, norfloxacin, ofloxacin, pefloxacin), aminoglycosides (amikacin, gentamicin, netilmicin, streptomycin, tobramycin), β-lactams (aztreonam, ceftazidime, imipenem, ticarcilin) and macrolides (erythromycin, roxitromycin) on the excretion of alginate by aP. aeruginosa strain were studied. Both β-lactam and macrolide antibiotics were found ineffective at the concentrations tested, except erythromycin and imipenem at 1/4 MIC. Aminoglycosides at a concentration of 1/4 MIC reduced most effectively the excretion of alginate. Quinolones were also effective at this sub-MIC; 1/16 MIC was ineffective with all antibiotics or stimulated the production of alginate.     

Effect of supplementation with exogenous fatty acid on the biological properties of a fatty acid requiring auxotroph ofSalmonella typhimurium

The effects of changes in fatty acid composition of the cell membrane on different biological functions ofSalmonella typhimurium have been studied with the help of a temperature sensitive fatty acid auxotroph which cannot synthesise unsaturated fatty acids at high temperature. On being shifted to nonpermissive temperature the cells continue growing for another one and half to two generations. The rates of protein and DNA syntheses run parallel to the growth rate but the rate of RNA synthesis is reduced. Further, there is a gradual reduction in the rate of transport of exogenous uridine and thymidine into the soluble pool. The transport process can be restored by supplementing the growth medium with cis-unsaturated fatty acids but not trans-unsaturated ones although the growth of the cells is resumed by supplementation with eithercis or trans-unsaturated fatty acids. However, supplementation withtrans, trans-unsaturated fatty acids leads to only partial recovery of the transport process. The rate of oxygen uptake is also affected in cells grown in the presence of thetrans-unsaturated fatty acids, elaidic acid and palmitelaidic acid. Analysis of cells grown under different fatty acid supplementation indicate that fatty acid composition of the cell membrane, especially the ratio of unsaturated to saturated fatty acids varies with temperature shift and supplementation of the growth media with fatty acids.     

Effect of subinhibitory concentrations of antibiotics on catalase activity of plague microbes]

It was shown that the presence of subinhibitory concentrations of ampicillin, cefotaxime or gentamicin in the cultivation medium had a marked inhibitory effect on the catalase activity of plague microbe. The effect depended on the characteristic features of plague microbe strains and the incubation temperature. When the cells of a virulent strain of the plague microbe Y. pestis 1300 were cultivated at a temperature of 37 degrees C on a medium containing the subinhibitory concentrations of ampicillin or cefotaxime, the pathogen virulence for albino mice significantly decreased.     

Effect ofcis andtrans unsaturated fatty acids on the transport properties ofSalmonella typhimurium

The transport of α-methyl-D-glucoside and two aminoacids, L-phenylalanine and L-leucine by a temperature sensitive fatty acid requiring mutant ofSalmonella typhimurium was studied under conditions of supplementation withcis or trans-unsaturated fatty acids. The results of such experiments definitely establish a relationship between the fatty acids composition of the membrane and the transport property of the cells. Cells grown in the presence of trans-unsaturated fatty acids cannot transport so efficiently as compared to the cis-unsaturated fatty acid-grown cells except linolelaidic acid, atrans-trans-unsaturated fatty acid. Protein: phospholipid ratio of the membrane also varies significantly under such conditions. The affinity of L-phenylalanine transport carrier for the substrate changes remarkably in cells grown in the presence of differentcis or trans-unsaturated fatty acids and indicate the possible role of membrane lipids in membrane assembly as well as regulation of the activity of L-phenylalanine transport system.     

Postantibiotic effects of subinhibitory concentrations of some antibiotics and their influence onPseudomonas aeruginosa enzymic activity

The postantibiotic effects of subinhibitory concentrations (PA SMEs) and virulence factor alterations induced by ciprofloxacin, tobramycin and netilmicin inPseudomonas aeruginosa were studied. After induction of the postantibiotic phase (PA) (2x or 4x MIC) the cultures were exposed to subinhibitory concentrations (0.1, 0.2 and 0.3x MIC) of the same antibiotic PA SME). The regrowth of treated as well as control cultures was followed for 24 or 45 h. In the sterile culture filtrates obtained from these bacterial cultures, elastase and proteinase were determined. Ciprofloxacin and aminoglycosides exhibited PA SMEs of 3.5–35 h for certain combinations of supra-subinhibitory antibiotic concentrations. Longer PA SMEs were observed after treatment with higher sub-MICs. Tobramycin at 0.2 and 0.3x MIC (postantibiotic phase induced by 2x MIC) and at all sub-MICs added to the bacteria previously exposed to 4x MIC do not allow any regrowth of bacterial culture. PA SMEs of tested antibiotics affected virulence factors ofP. aeruginosa. Elastase compared to proteinase was suppressed more effectively. Ciprofloxacin at 0.3x MIC reduced elastase and proteinase activity most significantly (to 14.2 and 60 % of the control values).     

Influence of lipopolysaccharide on external hemolytic activity ofSalmonella typhimurium andKlebsiella pneumoniae

Excretion ofEscherichia coli -hemolysin was tested in rough and smooth strains ofKlebsiella pneumoniae andSalmonella typhimurium. Smooth strains harboring the hemolytic recombinant plasmid pANN202-312 showed a five- to tenfold increase in the hemolytic activity evaluated in the external medium compared with isogenic rough strains harboring the same plasmid.     

Anionic sites on the envelope ofSalmonella typhimurium mapped with cationized ferritin

Binding of either ferritin (F) or cationized ferritin (CF) was employed to indicate the surface charge of the envelope of mainly twoSalmonella typhimurium strains (395 MR10, a Rd-mutant, and LT2-M1, a UDP-galactose-4-epimerase-less mutant). Lowering the pH from 7 to 4 decreased binding of CF, but increased binding of F. At low concentrations, the distribution of CF onS. typhimurium 395 MR10 was in general random, with individual ferritin molecules often forming clusters of two or three particles. At ionic strengths of 0.25M NaCl, ferritin produced distinctive, larger clusters at relatively few sites (10–50/cell). Addition of galactose to cultures of growingS. typhimurium, LT2-M1 reduced the binding of CF in 1–10 min, and numerous ferritinfree areas became visible. Possibly this is caused by a pluri-focal reduction in the negative cell surface charge that was generated at the multiple sites of export of new, smooth-type lipopolysaccharide, which either exhibits lesser charge or masks a preexisting surface charge. Dividing cells may show unequal charges on the prospective daughter cells, and the difference in the capacity for ferritin adsorption of both daughter cells is sharply separated at the division site.     

A genetical and biochemical study of chlorate-resistant mutants ofSalmonella typhimurium

S.typhimurium can form nitrate reductase A, chlorate reductase C, thiosulfate reductase, tetrathionate reductase and formic dehydrogenase. None of these enzymes are formed in chlorate-resistant mutants. Conjugation experiments showed the presence of a strong linkage between thechl andgal markers of the bacterial chromosome. By deletion mapping the gene ordernic A aro G gal bio chl D uvr B chl A was found. Strains with deletions terminating betweenbio anduvr B or betweenuvr B andchl A have a number of aberrant properties. Though resistant against chlorate they reduce nitrate and form gas. After growth with nitrate they form less nitrate reductase than the wild type which may explain the resistance against chlorate. After growth with thiosulfate they form small amounts of thiosulfate reductase and chlorate reductase C. In crosses between anE.coli Hfrchl ⁺ strain and aS.typhimurium chl A strain recombinants were obtained, forming nitrate reductase A and chlorate reductase C. These recombinants do not form gas, which indicates that thechl ⁺ gene fromE.coli does not function normally inS.typhimurium.The author is very gratefull to Miss C. W. Bettenhaussen, Miss W. M. C. Kapteijn and Mr. K. Pietersma for technical assistance. Helpfull suggestions of Dr. P. van de Putte (Medical Biological Laboratory of the National Defence Organization TNO, Rijswijk) are gratefully acknowledged.     

Effect of subinhibitory concentrations of antimicrobials on meningococcal adherence

Meningococci adhere to human pharyngeal cells and agglutinate erythrocytes. These events are dependent upon pili and are reduced by encapsulation. The effect of subinhibitory concentrations of seven antimicrobials on meningococcal adherence, antimicrobials on meningococcal adherence, piliation, hemagglutination (HA), and bacterial proteins was studied to determine their potential for modifying virulence. Piliation was reduced by most antibiotics but was most markedly (greater than 70%) reduced by rifampin, tobramycin, and VCN (vancomycin, colistin, and nystatin). Bacterial proteins as determined by sodium dodecyl sulphate--polyacrylamide gel electrophoresis were altered: tetracycline, sulfamethoxazole, rifampin, and VCN caused loss of a 43-45 K protein and a general decrease in all stainable protein bands, while erythromycin, ampicillin, and tobramycin only caused an increase in a 28 K protein. HA was reduced by ampicillin, tobramycin, erythromycin, and VCN but interstrain variability was present. Epithelial cell adherence was diminished by an average of 45% compared to controls. The meningococcal strains lost HA, piliation, and adherence in the same rank order, however, there was no significant rank correlation of antibiotic inhibitory activities on these parameters. These results indicate that subinhibitory antibiotic concentrations reduce meningococcal piliation and alter other bacterial proteins; these changes are associated with diminished adherence and hemagglutination, alterations which may be markers of meningococcal virulence.     

Sensitivity of bacteria exposed to subinhibitory concentrations of antibiotics to the action of serum and liver phagocytes

Incubation of E.coli and S. aureus with subinhibitory concentration (1/5 MIC) of cefamandole modified bacterial morphology and resistance to host defence mechanisms. In fact, cefamandole induced filamentous forms of E. coli, when added to the perfusing medium of the isolated rat liver system, were phagocytized at a much fortes rate then control bacteria, but appeared less sensitive to serum bactericidal activity. In contrast, S.aureus, after exposure to the antibiotic, was more sensitive to the bactericidal activity of serum then controls, while treated and untreated cells were phagocytized at the some rate. The data suggest that even at low doses some antibiotics may alter bacterial structure and increase their susceptibility to host factors.     

Comparative evaluation of biological properties of Salmonella typhimurium strains of different origin and genetic nature of resistance to antibiotics

The results obtained in the comparative study of the biological characteristics of Salmonella typhimurium strains of different origin are presented. The circulation of two biovariants differing in a number of biological characteristics, mainly in their susceptibility to antibiotics, has been shown. R-plasmids, mostly with the markers of resistance to tetracycline and with a molecular weight of 64 Md, have been isolated from "hospital" and "sporadic" strains possessing multiple antibiotic resistance.     

Constancy of diameter through the cell cycle ofSalmonella typhimurium LT2

Measurements of cell diameter and length inSalmonella typhimurium LT2 cells were correlated using both light and electron microscopy. In cultures growing at high, intermediate, and low rates, cell diameter does not change with length. This constancy is also maintained in septated cells before division. Since length increases continuously with cell age, the above observations mean that cells maintain a constant diameter during the cell cycle.     

Association with HeLa cells of LPS mutants ofSalmonella typhimurium andSalmonella minnesota in relation to their physicochemical surface properties

Different LPS mutants ofSalmonella typhimurium andSalmonella minnesota have been investigated with respect to (1) their tendency to associate, with HeLa cell monolayers, and (2) their physicochemical surface properties. Aqueous biphasic partitioning, hydrophobic interaction chromatography, and ion exchange chromatography have been used to characterize the bacterial cell surface properties with respect to charge and hydrophobicity. Liability to hydrophobic interaction was defined either by the change of partition in a dextran-polyethylene-glycol (PEG) system by the addition of PEG-palmitate (P-PEG), or by the elution pattern from Octyl-Sepharose. Accordingly, charge was assessed by the effect of positively charged trimethylamino-PEG (TMA-PEG) on the partition, and by the elution from DEAE-Sephacel. Bacterial being negatively charged and liable to hydrophobic interaction had the highest tendency to associate with HeLa cells. In some cases the methods for surface analysis gave conflicting results on charge and/or liability to hydrophobic interaction of the same LPS mutant. Possible reasons for these differences and the role of bacterial cell surface structures contributing to physicochemical character are discussed.     

Antimutagenic effects of caffeine during nitrosoguanidine-induced mutagenesis ofSalmonella typhimurium cells and phages

The effect of caffeine on nitrosoguanidine-induced mutagenesis ofSalmonella typhimurium & nd its P22 and L phages was studied. The detected mutations included phage “clear” mutations, reversions of phage “amber” mutation, and prototrophic reversions of thehis ⁻ auxotroph ofSalmonella typhimurium. Neither therecA mutation of the host nor theerf mutation of the phage genome were found to affect the nitrosoguanidine-induced mutagenesis of the phage during vegetative growth. Beginning with a concentration of 0.2 mg/ml, caffeine decreased the frequency of mutants by 30–60%, attaining a maximum effect at 1.5 mg/ml and retaining this effect even at higher concentrations. A similar antimutagenic effeot was observed with the mutagenesis of the host cells. The nitrosoguanidine-induced mutagenesis does not seem to be related to the function of therecA cell gene or theerf phage gene. The mechanism of mutagenesis by nitrosoguanidine probably has two components, one of them caffeine sensitive, the other caffeine-resistant.