Assays optimized for detection and quantification of antibacterial activity in shark cell lysates under high salt conditions

To assess non-cellular innate immune mechanisms that play a role in the antimicrobial defense of an organism several assay systems have been devised to screen for such factors. Most assays, however, have been developed to measure activity against clinical isolates of medical importance. There is scant information on methods optimal for assaying material from sharks and other marine fish for antimicrobial activity particularly against salt tolerant organisms that are likely to be encountered in the marine environment. We have modified and optimized agar diffusion and broth dilution assays for detection and quantification of antibacterial activity of shark leukocyte lysates. By replacing marine agar, typically used for marine organisms, with artificial sea water complete medium (SCM) enriched with tryptone and yeast extract has resulted in an improved inhibition zone assay that uses Planococcus citreus, a salt-tolerant organism as the target organism. Antibacterial activity is correlated to the size of zone of no bacterial growth around wells containing bioactive test sample. An alternative broth based microdilution growth assay uses the 96 well format and the antibacterial effect of the sample on growth of P. citreus, the target organism, is measured spectrophotometrically as percent inhibition of bacterial growth when compared to the growth of P. citreus grown in medium alone that represents 100% growth. The assay can also be used to titrate antibacterial activity and express the level of growth inhibition as a titer.     

Elucidation of Growth Inhibition and Acetic Acid Production by Clostridium thermoaceticum

The production of acetic acid by Clostridium thermoaceticum was studied by using batch fermentations. In a pH-controlled fermentation with sodium hydroxide (pH 6.9), this organism was able to produce 56 g of acetic acid per liter. On the other hand, when the pH was not controlled and was decreased during fermentation to 5.4, the maximum attainable acetic acid concentration was only 15.3 g/liter. To obtain a better understanding of the end product inhibition, various salts were tested to determine their effect on the growth rate of C. thermoaceticum. An inverse linear relationship between the growth rate and the final cell concentration to the sodium acetate concentration was found. By using different concentrations of externally added sodium salts, the relative growth inhibition caused by the anion was found to be in the order of acetate > chloride > sulfate. Various externally added cations of acetate were also examined with respect to their inhibitory effects on growth. The relative magnitude of inhibition on the growth rate was found to be ammonium > potassium > sodium. The combined results have shown that the undissociated acetic acid was much more inhibitory than the ionized acetate ion. Complete growth inhibition resulted when the undissociated acetic acid concentration was between 0.04 and 0.05 M and when the ionized acetate concentration was 0.8 M. Therefore, at low pH (below 6.0), undissociated acetic acid is responsible for growth inhibition, and at high pH (above 6.0), ionized acetate ion is responsible for growth inhibition.     

A PRELIMINARY BACTERIOLOGICAL SCREENING TEST FOR QUATERNARY AMMONIUM COMPOUNDS AND FORMULATIONS

SUMMARY: The test is based on a colony count method to determine the survival of a given test organism, Bacterium coli , in the presence of sterile homogenized whole milk added with the bacterial suspension to the quaternary ammonium compound dilutions, for an exposure time of two min.
Attention is drawn to the special cleaning of the glassware used. The preparation and use of the Q.A.C. inhibitor, lecithin in 'Lissapol N', is described, with details of the testing procedure.
Inconsistencies in survivor counts occurring when Q.A.C. solutions were tested against saline suspensions of Bact. coli were reduced by carrying out the test in the presence of whole milk solids.
Examples quoted show how this test indicated a difference in bactericidal efficiency between (a) two different Q.A.C. solutions, and (b) the same Q.A.C. included in two powder formulations of differing pH value.     

fus-1, a pH Shift Mutant of Semliki Forest Virus, Acts by Altering Spike Subunit Interactions via a Mutation in the E2 Subunit

Semliki Forest virus (SFV), an enveloped alphavirus, is a well-characterized paradigm for viruses that infect cells via endocytic uptake and low-pH-triggered fusion. The SFV spike protein is composed of a dimer of E1 and E2 transmembrane subunits, which dissociate upon exposure to low pH, liberating E2 and the fusogenic E1 subunit to undergo independent conformational changes. SFV fusion and infection are blocked by agents such as ammonium chloride, which act by raising the pH in the endosome and inhibiting the low-pH-induced conformational changes in the SFV spike protein. We have previously isolated an SFV mutant, fus-1, that requires more acidic pH to trigger its fusion activity and is therefore more sensitive to inhibition by ammonium chloride. The acid shift in the fusion activity of fus-1 was here shown to be due to a more acidic pH threshold for the initial dissociation of the fus-1 spike dimer, thereby resulting in a more acidic pH requirement for the subsequent conformational changes in both fus-1 E1 and fus-1 E2. Sequence analysis demonstrated that the fus-1 phenotype was due to a mutation in the E2 spike subunit, threonine 12 to isoleucine. fus-1 revertants that have regained the parental fusion phenotype and ammonium chloride sensitivity were shown to have also regained E2 threonine 12. Our results identify a region of the SFV E2 spike protein subunit that regulates the pH dependence of E1-catalyzed fusion by controlling the dissociation of the E1/E2 dimer.     

Antibacterial protection in Marthasterias glacialis eggs: characterization of lysozyme-like activity

Eggs from Marthastherias glacialis exert antibacterial action on marine bacterial strains and show a lysozyme-like activity. This one depends on pH and ionic strength of sample and reacting medium. This hydrolase, purified by gel filtration and ion-exchange chromatography, could be responsible for the bacterial growth inhibitory activity observed.     

Paraformaldehyde-Resistant Starch-Fermenting Bacteria in “Starch-Base” Drilling Mud

Starch-fermenting bacteria were found in each of 12 samples of nonfermenting starch-base drilling mud examined. Of the 12 samples, 3 contained very active starch-fermenting gram-positive spore-bearing bacilli closely resembling Bacillus subtilis. Similar active starch-fermenting bacteria were found in fermenting starch-base drilling mud and in corn starch and slough water used to prepare such mud. The active starch-fermenting microorganisms completely hydrolyzed 1% (w/v) corn starch within 24 hr at 37.5 C. The active starch-fermenting bacteria isolated from fermenting drilling mud were capable of surviving 12 hr of continuous exposure to 0.1% (w/w) paraformaldehyde or 1 hr of continuous exposure to 0.5% (w/w) paraformaldehyde, with no diminution in starch-fermenting ability. The same organisms fermented starch after 3 hr of continuous exposure to 0.5% (w/w) paraformaldehyde, but not after 4 hr of exposure. The phenomenon of rapid disappearance of paraformaldehyde from fermenting drilling mud was observed in the laboratory using a modified sodium sulfite test. Paraformaldehyde, initially present in a concentration of 0.192 lb per barrel of mud, completely disappeared in 9 hr at 22 to 23 C. A significant decrease in paraformaldehyde concentration was detected 0.5 hr after preparation of the mud. It is suggested that the presence of relatively high concentrations of ammonia and chloride in the mud may facilitate the disappearance of paraformaldehyde. The failure of 0.1% (w/w) paraformaldehyde to inhibit the strong starch-fermenting microorganisms isolated from fermenting drilling mud, and the rapid disappearance of paraformaldehyde from the mud, explains the fermentation of starch which occurred in this mud, despite the addition of paraformaldehyde.     

Etude de l'activite de l'arginine decarboxylase dans les jeunes plantes de Glycine max privees de leurs cotyledons

l-Arginine carboxy-lyase and l-ornithine carboxy-lyase activities were investigated in young decotylized Glycine max plants growing in the light on a liquid medium in the presence of ammonium chloride or nitrate. Only the first enzyme could be detected. It had pH optimum at 7.0 activity and there is much more activity in plants cultivated on ammonium chloride than in plants grown on nitrate.     

Bactericidal activity of human peritoneal macrophages from patients undergoing peritoneal dialysis

Peritoneal macrophages (PM) play an essential role in the pathogenesis of bacterial peritonitis, the main complication of peritoneal dialysis (PD). We determined the antibacterial activity of PM from 31 PD patients using gram-positive (Staphylococcus aureus, Staphylococcus epidermidis) and gram-negative (Escherichia coli, Pseudomonas aeruginosa) test organisms. In an 8-hour test assay, PM revealed the highest antibacterial activity against E. coli [median bactericidal index (Bi) = 5.46 representing 0.74 log growth inhibition compared to controls] and the lowest against P. aeruginosa (Bi = 1.63, 0.21 log growth inhibition, p less than 0.05). The antibacterial activity against S. aureus (Bi = 1.99, 0.3 log growth inhibition) and S. epidermidis (Bi = 2.0, 0.31 log growth inhibition) was within this range. When compared to peripheral blood polymorphonuclear leukocytes, PM reached only 4% (S. aureus) and 8.1% (E. coli) of their antibacterial activity (p less than 0.05). Using E. coli as a test organism, PM isolated after a 4-hour dialysis period revealed the highest antibacterial activity when compared to PM isolated after longer dialysis periods (p less than 0.05). Increasing the duration of PD to 6 and 8 h subsequently decreased the antibacterial activity of PM, suggesting that unphysiologic concentrations of toxic metabolites in the peritoneal effluent might have a harmful influence on PM functions.     

Properties of in vivo nitrogenase activity in Beggiatoa alba

A procedure was devised for analyzing in vivo nitrogenase activity in Beggiatoa alba B18LD which involves: (1) the induction of nitrogenase in cells pre-grown on NH₄Cl, by washing the cells free of NH₄Cl and lowering their exposure to oxygen, and (2) measuring acetylene reduction by these cells. Using this induction methodology we examined the effects of pH, temperature, and nitrogenous compounds on in vivo nitrogenase induction and activity in Beggiatoa alba B18LD. Nitrate and nitrite repressed the induction of nitrogenase activity, but glutamine did not. Induction and activity had a combined pH optimum of 6.5 to 8.0, and activity had a temperature optimum of 29°C. Ammonium and urea caused immediate inhibition of nitrogenase activity, but nitrate, nitrite, glutamine, asparagine, and other amino acids did not. Ammonium-induced inhibition was transient and incomplete, and the duration of inhibition increased in direct proportion to the amount of ammonium added. Methionine sulfoximine, a glutamine synthetase inhibitor, at a final concentration of 50 μM blocked ammonium uptake by cells, but did not prevent nitrogenase inhibition if added before ammonium. Our results imply that B. alba nitrogenase inhibition by ammonium: (1) is not directly caused by ammonium assimilation products, (2) is probably not due to an enzymatic inactivation, and (3) may be related to ammonium transport.     

Synthesis, characterization, and antibacterial activity of N,O-quaternary ammonium chitosan

N,N,N-Trimethyl O-(2-hydroxy-3-trimethylammonium propyl) chitosans (TMHTMAPC) with different degrees of O-substitution were synthesized by reacting O-methyl-free N,N,N-trimethyl chitosan (TMC) with 3-chloro-2-hydroxy-propyl trimethyl ammonium chloride (CHPTMAC). The products were characterized by ¹H NMR, FTIR and TGA, and investigated for antibacterial activity against Staphylococcus aureus and Escherichia coli under weakly acidic (pH 5.5) and weakly basic (pH 7.2) conditions. TMHTMAPC exhibited enhanced antibacterial activity compared with TMC, and the activity of TMHTMAPC increased with an increase in the degree of substitution. Divalent cations (Ba²⁺ and Ca²⁺) strongly reduced the antibacterial activity of chitosan, O-carboxymethyl chitosan and N,N,N-trimethyl-O-carboxymethyl chitosan, but the repression on the antibacterial activity of TMC and TMHTMAPC was weaker. This indicates that the free amino group on chitosan backbone is the main functional group interacting with divalent cations. The existence of 100 mM Na⁺ slightly reduced the antibacterial activity of both chitosan and its derivatives.     

Changes in Sensitivity to Effectors of Maize Leaf Phosphoenolypyruvate Carboxylase during Light/Dark Transitions

Illumination of previously darkened maize (Zea mays L. cv Golden Cross Bantam T51) leaves had no effect on the concentration of phosphoenolpyruvate (PEP) carboxylase protein, but increased enzyme activity about 2-fold when assayed under suboptimal conditions (pH 7.0 and limiting PEP). In addition, sensitivity to effectors of PEP carboxylase activity was significantly altered; e.g. malate inhibition was reduced and glucose-6-phosphate activation was increased. Consequently, 10- to 20-fold differences in PEP carboxylase activity were observed during dark to light transitions when assayed in the presence of effectors. At pH 7.0 activity of purified PEP carboxylase was not proportional to enzyme concentrations. Below 0.7 microgram PEP carboxylase protein per milliliter, enzyme activity was disproportionately reduced. Including polyethylene glycol plus potassium chloride in the reaction mixture eliminated this discontinuity and substantially increased PEP carboxylase activity and reduced malate inhibition dramatically. Inclusion of polyethylene glycol in the assay mixture specifically increased the activity of PEP carboxylase extracted from dark leaves, and reduced malate inhibition of the enzyme from both light and dark leaves. Collectively, the results suggest that PEP carboxylase in maize leaves is subjected to some type of protein modification that affects both activity and effector sensitivity. We postulate that changes in quaternary structure (dissociation or altered subunit interactions) may be involved.     

Purification and Some Biological Properties of Asparaginase from Azotobacter vinelandii

Asparaginase was found in the soluble fraction of cells of Azotobacter vinelandii, and its activity remained the same during growth of the organism in a nitrogen-free medium. The specific activity and the yield of A. vinelandii increased twofold in the presence of ammonium sulfate. Within limits, the temperature (30 to 37°C) and pH (6.5 to 8.0) of the medium showed little effect on the levels of enzyme activity. The enzyme was purified to near homogeneity by standard methods of enzyme purification, including affinity chromatography, and had optimum activity at pH 8.6 and 48°C. The approximate molecular weight was 84,000. The apparent K_m value for the substrate was 1.1 × 10^-4 M. Metal ions or sulfhydryl reagents were not required for enzyme activity. Cu²⁺, Zn²⁺, and Hg²⁺ showed concentration-dependent inhibition, whereas amino and keto acids had no effect on the enzyme activity. Asparaginase was stable when incubated with rat serum and ascites fluid. The enzyme had no effect on the membrane of sheep erythrocytes and did not inhibit the incorporation of radioactive precursors into deoxyribonucleic acid, ribonucleic acid, and protein in Yoshida ascites sarcoma cells. Asparaginase activity was not detected in the tumor cells.     

Growth, nitrogen utilization and biodiesel potential for two chlorophytes grown on ammonium, nitrate or urea

Nitrogen removal from wastewater by algae provides the potential benefit of producing lipids for biodiesel and biomass for anaerobic digestion. Further, ammonium is the renewable form of nitrogen produced during anaerobic digestion and one of the main nitrogen sources associated with wastewater. The wastewater isolates Scenedesmus sp. 131 and Monoraphidium sp. 92 were grown with ammonium, nitrate, or urea in the presence of 5 % CO₂, and ammonium and nitrate in the presence of air to optimize the growth and biofuel production of these chlorophytes. Results showed that growth on ammonium, in both 5 % CO₂ and air, caused a significant decrease in pH during the exponential phase causing growth inhibition due to the low buffering capacity of the medium. Therefore, biological buffers and pH controllers were utilized to prevent a decrease in pH. Growth on ammonium with pH control (synthetic buffers or KOH dosing) demonstrated that growth (rate and yield), biodiesel production, and ammonium utilization, similar to nitrate- and urea-amended treatments, can be achieved if sufficient CO₂ is available. Since the use of buffers is economically limited to laboratory-scale experiments, chemical pH control could bridge the gap encountered in the scale-up to industrial processes.     

Stimulation of tyrosinase activity of cultured melanoma cells by lysosomotropic agents

The tyrosinase (EC 1.14.18.1) activity of cell-free extracts (TyH) of B16 melanoma cells cultured in the presence of 5 to 10 mM ammonium chloride was considerably higher than that of cells from control cultures. This increase in TyH in the presence of ammonium chloride seemed to be due to de novo synthesis of the enzyme, because it was inhibited by 1 microgram/ml of cycloheximide. In the presence of the latter, however, ammonium chloride did increase the tyrosinase activity of living cells in culture (TyC) resulting in about threefold increase in the TyC/TyH ratio, a measure of the extent of tyrosinase reaction exerted by the enzyme present in living cells. This higher TyC/TyH ratio induced by ammonium chloride was also observed in the absence of cycloheximide. Similar increases in TyH, TyC, and TyC/TyH occurred in the presence of methylamine or ethylamine instead of ammonium chloride, but not in the presence of tetraethylammonium chloride, and also in culture medium of higher pH. The apparently similar effects of lysosomotropic bases and medium of higher pH on the TyC/TyH ratio suggest that there are some mechanisms that control the intramelanosomal pH lower than the cytoplasmic pH.     

Quaternization of N-aryl chitosan derivatives: synthesis, characterization, and antibacterial activity

Chemical modification of chitosan by introducing quaternary ammonium moieties into the polymer backbone renders excellent antimicrobial activity to the adducts. In the present study, we have synthesized 17 derivatives of chitosan consisting of a variety of N-aryl substituents bearing either electron-donating or electron-withdrawing groups. Selective N-arylation of chitosan was performed via Schiff bases formed by the reaction between the 2-amino groups of the glucosamine residue of chitosan with aromatic aldehydes under acidic conditions, followed by reduction of the Schiff base intermediates with sodium cyanoborohydride. Each of the derivatives was further quaternized using N-(3-chloro-2-hydroxypropyl)trimethylammonium chloride (Quat-188) as the quaternizing agent that reacted with either the primary amino or hydroxyl groups of the glucosamine residue of chitosan. The resulting quaternized materials were water soluble at neutral pH. Minimum inhibitory concentration (MIC) antimicrobial studies of these materials were carried out on Escherichia coli (Gram-negative) and Staphylococcus aureus (Gram-positive) bacteria in order to explore the impact of the extent of N-substitution (ES) on their biological activities. At ES less than 10%, the presence of the hydrophobic substituent, such as benzyl and thiophenylmethyl, yielded derivatives with lower MIC values than chitosan Quat-188. Derivatives with higher ES exhibited reduced antibacterial activity due to low quaternary ammonium moiety content. At the same degree of quaternization, all quaternized N-aryl chitosan derivatives bearing either electron-donating or electron-withdrawing substituents did not contribute antibacterial activity relative to chitosan Quat-188. Neither the functional group nor its orientation impacted the MIC values significantly.     

Mode of inhibition of diphtheria toxin by ammonium chloride

Kim, K. (University of Washington, Seattle), and N. B. Groman. Mode of inhibition of diphtheria toxin by ammonium chloride. J. Bacteriol. 90:1557-1562. 1965.-The inhibition of diphtheria toxin by ammonium salts was independent of toxin concentration over a 100-fold range of toxin. Inhibition by minimal concentrations of ammonium chloride was abolished by lowering the pH, indicating that free ammonia is the active form of inhibitor. A single addition of ammonium chloride inhibited toxin for a limited period of time, but periodic readdition of the ammonium salt was required to sustain inhibition indefinitely in the absence of antitoxin. Toxin was not destroyed and its adsorption occurred equally well in the presence or absence of ammonium chloride. Preadsorbed toxin was also effectively inhibited by the addition of ammonium chloride. Inhibited toxin remained accessible to antitoxin neutralization. Attempts to reverse ammonia inhibition by the addition of succinate or reduced nicotinamide adenine dinucleotide were unsuccessful. Attempts to inhibit toxin by interfering with active transport were also unsuccessful.     

Controlled synthesis of Mn2O3 nanowires by hydrothermal method and their bactericidal and cytotoxic impact: a promising future material

Mn?O? nanowires with diameter ~70 nm were synthesized by a simple hydrothermal method using Mn(II) nitrate as precursor. X-ray diffraction, field emission scanning electron microscopy, transmission electron microscopy (TEM), and energy dispersive X-ray spectroscopy techniques were employed to study structural features and chemical composition of the synthesized nanowires. A biological evaluation of the antimicrobial activity and cytotoxicity of Mn?O? nanowires was carried out using Escherichia coli and mouse myoblast C?C?? cells as model organism and cell lines, respectively. The antibacterial activity and the acting mechanism of Mn?O? nanowires were investigated by using growth inhibition studies and analyzing the morphology of the bacterial cells following the treatment with nanowires. These results suggest that the pH is critical factor affecting the morphology and production of the Mn?O? nanowires. Method developed in the present study provided optimum production of Mn?O? nanowires at pH?~?9. The Mn?O? nanowires showed significant antibacterial activity against the E. coli strain, and the lowest concentration of Mn?O? nanowires solution inhibiting the growth of E. coli was found to be 12.5 μg/ml. TEM analysis demonstrated that the exposure of the selected microbial strains to the nanowires led to disruption of the cell membranes and leakage of the internal contents. Furthermore, the cytotoxicity results showed that the inhibition of C?C?? increases with the increase in concentration of Mn?O? nanowires. Our results for the first time highlight the cytotoxic and bactericidal potential of Mn?O? nanowires which can be utilized for various biomedical applications.     

Effects of nitrate,ammonium and pH on the growth of conifer seedlings and their production of nitrate reductase

Summary Lodgepole pine (Pinus contorta Dougl.), Engelmann spruce (Picea engelmanni Parry), and Douglas-fir (Pseudotsuga menziesii (Mirb.) Franco) seedlings were grown in open-ended tube cultures of sand and perlite, irrigated with nitrate, ammonium, and a 1∶1 mixture of ammonium and nitrate, combined factorially with pH values of 4.6, 5.3 and 6.0 giving a total of nine treatments. Douglas-fir showed intolerance to ammonium which was especially marked in root weight. Lodgepole pine and Engelmann spruce made poor growth with nitrate, but showed little difference between ammonium and mixed sources. Only Douglas-fir showed a significant response to pH treatments with pH 5.3 plants being largest. Contamination of the sand with carbonate-bicarbonate, apparently caused seedlings grown in ammonium solutions to be larger in sand than in perlite. Douglas-fir grown in perlite cultures showed a growth response like the first experiment and nitrate reductase activity in the order nitrate > nitrateammonium mixture > ammonium. Plastic bead cultures had poor growth response due to low retention of water by the substrate, but the nitrate reductase assays produced results like the perlite cultures. Lodgepole pine grown in water culture demonstrated the well known pH shift associated with different nitrogen forms, and when assayed for nitrate reductase these seedlings had larger relative activities than Douglas-fir, but the order of activity remained nitrate > mixed source > ammonium.     

Nonleaching antimicrobial films prepared from surface-modified microfibrillated cellulose

We have prepared potentially permanent antimicrobial films based on surface-modified microfibrillated cellulose (MFC). MFC, obtained by disintegration of bleached softwood sulfite pulp in a homogenizer, was grafted with the quaternary ammonium compound octadecyldimethyl(3-trimethoxysilylpropyl)ammonium chloride (ODDMAC) by a simple adsorption-curing process. Films prepared from the ODDMAC-modified MFC were characterized by Fourier transform infrared spectroscopy (FT-IR) and X-ray photoelectron spectroscopy (XPS) and tested for antibacterial activity against the Gram-positive bacterium Staphylococcus aureus and the Gram-negative bacteria Escherichia coli and Pseudomonas aeruginosa. The films showed substantial antibacterial capacity even at very low concentrations of antimicrobial agent immobilized on the surface. A zone of inhibition test demonstrated that no ODDMAC diffused into the surroundings, verifying that the films were indeed of the nonleaching type.     

Identification and characterization of antimicrobial activity in two yeast genera

A general screening test for the expression of antibacterial activity was performed on over 400 cultures belonging to 31 yeast genera. Of these cultures, only two, Kluyveromyces thermotolerans and Kloeckera apiculata, were found to produce zones of inhibition of bacterial growth on Diagnostic Sensitivity Test Agar medium supplemented with 0.002% methylene blue. Of nine bacteria used as test organisms, only Lactobacillus plantarum and Bacillus megaterium were inhibited. No antibacterial activity was evident against four gram-negative bacteria used in this study. Optimal activities were found to be expressed after yeasts were grown at pH 6. A requirement for cultivation in the presence of methylene blue added to culture media for the expression of apparent antibacterial activity was demonstrated.