Influence of specific growth rate and nutrient limitation upon the sensitivity of Escherichia coli towards chlorhexidine diacetate

The sensitivity of Escherichia coli to chlorhexidine has been assessed for cells grown in a chemostat at a variety of specific growth rates, under conditions of carbon, nitrogen, phosphorus and magnesium limitation. At slow rates of growth (ca 0.08/h) little difference in sensitivity was observed. As growth rate was increased, however, the sensitivity of nitrogen- and carbon-limited cells increased whilst that of magnesium- and phosphate-limited cells decreased. It was not possible to correlate the observed patterns of chlorhexidine sensitivity with any single measure of cell envelope composition (phospholipid content, lipopolysaccharide, envelope proteins, etc.). The results presented are not consistent, therefore, with any simple model for chlorhexidine binding or action and more probably reflect subtle interaction between chlorhexidine, phospholipid-lipopolysaccharide complexes and cations within the envelope.     

Cell Wall-mediated Changes in Sensitivity of Bacillus megaterium to Chlorhexidine and 2-Phenoxyethanol, Associated with Growth Rate and Nutrient Limitation

The resistance of chemostat-grown cultures of Bacillus megaterium (asporogenous) to the bactericidal action of chlorhexidine and 2-phenoxyethanol varied with growth rate and nutrient-limitation. Phosphate-limited cultures (P-lim) showed little change in sensitivity to either drug with changes in growth rate. Magnesium-limited (Mg-lim) and carbon-limited (C-lim) cultures, however, increased in sensitivity to both agents as growth rate was increased from 0.13–0.45 h. Minimum lytic concentrations of the agents were not significantly different for protoplasts prepared from these suspensions except of Mg-lim when sensitivity to chlorhexidine increased with growth rate. Lysozyme sensitivity of the cells varied with growth rate and nutrient limitation. Results support the idea that in addition to other effects of growth rate and nutrient-limitation environmentally-induced changes in envelope structure and/or composition radically influenced penetration of these agents to their targets.     

The effects of chlorhexidine on the maximum specific growth rate, biomass and hydrolytic enzyme production of Bacteroides gingivalis grown in continuous culture

Bacteroides gingivalis was grown in continuous culture in the presence of chlorhexidine. Maximum specific growth rates and biomass levels initially increased but then decreased as the chlorhexidine level increased from 0 to 30 micrograms/ml. Total inhibition of growth occurred when the chlorhexidine concentration reached 60 micrograms/ml. The steady-state levels of cell-bound, extracellular vesicle and extracellular soluble enzymes, trypsin-like protease, alkaline phosphatase and N-acetyl-beta-glucosaminidase were measured. With increasing sub-lethal concentrations of chlorhexidine, levels of alkaline phosphatase increased noticeably in all three fractions of culture, whilst cell-bound and extracellular vesicle levels of N-acetyl-beta-glucosaminidase remained approximately constant. Extracellular soluble levels of alkaline phosphatase and N-acetyl-beta-glucosaminidase increased with increasing levels of chlorhexidine. The levels of trypsin-like protease decreased significantly in all fractions of the culture when cells were grown in the presence of chlorhexidine. Thus, chlorhexidine has a differential effect on the production of B. gingivalis hydrolytic enzymes.     

Ultrastructural Study of Salmonella typhimurium Treated with Membrane-Active Agents: Specific Reaction of Dansylchloride with Cell Envelope Components

Amino groups of cell envelope proteins, lipids, and lipopolysaccharides cannot be labeled in intact cells of Salmonella typhimurium G 30 by using 5-dimethylaminonaphthalene-1-sulfonylchloride incorporated in lecithin-cholesterol vesicles. However, application of membrane-interacting agents like tris(hydroxymethyl)aminomethane (Tris)-hydrochloride, ethylenediaminetetraacetate (Na salt) (EDTA), divalent cations, and sublethal doses of the cationic antibacterial agents polymyxin B and chlorhexidine induced specific fluorescent labeling of envelope proteins and lipids but not of cytoplasmic compounds, with the exception of a soluble protein with a molecular weight of 46,000 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Treatment with Tris-hydrochloride buffer produced labeling of the heat-modifiable protein B/B(+) and of proteins with molecular weights of 26,000, 22,000, and below 17,000. A combination of Tris-hydrochloride and EDTA induced additional dansylation of the major protein A and of proteins of molecular weights 80,000, 60,000, and 44,000. Polymyxin B and chlorhexidine caused similar labeling patterns. In every case, except with divalent cation treatment, protein B/B(+) was the most prominently labeled species. Phosphatidylethanolamine was dansylated up to 30%. Lipopolysaccharide was not reactive under any condition or treatment. In addition, the peptidoglycan-bound lipoprotein did not react with dansylchloride in either intact or Tris-hydrochloride-treated cells. The results are discussed with regard to a possible localization of labeled and unlabeled compounds of the cell envelope on the basis of a model placing cell envelope amino groups into ion-ion interactions with anionic components of other envelope compounds like phosphate and carboxyl groups.     

The effects of chlorhexidine on the maximum specific growth rate, biomass and hydrolytic enzyme production of Bacteroides gingivalis grown in continuous culture

Bacteroides gingivalis was grown in continuous culture in the presence of chlorhexidine. Maximum specific growth rates and biomass levels initially increased but then decreased as the chlorhexidine level increased from 0 to 30 μg/ml. Total inhibition of growth occurred when the chlorhexidine concentration reached 60 μg/ml. The steady-state levels of cell-bound, extracellular vesicle and extracellular soluble enzymes, trypsin-like protease, alkaline phosphatase and N -acetyl-β-glucosaminidase were measured. With increasing sub-lethal concentrations of chlorhexidine, levels of alkaline phosphatase increased noticeably in all three fractions of culture, whilst cell-bound and extracellular vesicle levels of N -acetyl-β-glucosaminidase remained approximately constant. Extracellular soluble levels of alkaline phosphatase and N -acetyl-β-glucosaminidase increased with increasing levels of chlorhexidine. The levels of trypsin-like protease decreased significantly in all fractions of the culture when cells were grown in the presence of chlorhexidine. Thus, chlorhexidine has a differential effect on the production of B. gingivalis hydrolytic enzymes.     

Influence of Growth Rate and Nutrient Limitation on the Gross Cellular Composition of Pseudomonas aeruginosa and Its Resistance to 3- and 4-Chlorophenol

Concentrations of 3-chlorophenol and 4-chlorophenol below their minimal inhibitory concentrations were found to increase the permeability of Pseudomonas aeruginosa cells to protons. Levels of such activity were assessed in suspensions of cells prepared from chemostat-grown cultures, limited by either magnesium (Mg-lim) or glucose (G-lim), with the use of five growth rates. Drug concentrations required to produce the same levels of proton translocation varied with the growth rate and the nutrient limiting growth. Fast-growing cultures were more sensitive than slower-growing ones, and G-lim cells were generally more sensitive than Mg-lim ones. 3-Chlorophenol had greater activity than 4-chlorophenol at slow growth rates, but at faster rates of growth their activity was similar. Variation in these iso-effective concentrations for different cells probably reflected an alteration in the ease of drug penetration of the outer envelope. Uptake of the compounds by cells in suspension varied, drug-sensitive bacteria absorbing more than resistant ones. This variation in uptake persisted when bacteria were solvent-extracted to remove readily extractable lipids (REL). Since no significant alteration in cell size was observed among the growth conditions studied, variation in absorption probably resulted from an altered affinity of the cells to the drug, with little involvement of REL. Overall REL content did not alter significantly with growth rate or nutrient limitation. However, total phospholipid content decreased and fatty acid content increased with increasing growth rate. For G-lim and Mg-lim cultures phosphatidylcholine content remained constant, yet phosphatidylethanolamine and phosphatidylglycerol content decreased with increasing growth rate. Diphosphatidylglycerol content decreased with increasing growth rate for Mg-lim cultures and remained relatively constant for G-lim cultures. Lipopolysaccharide content of the cells was higher in Mg-lim than in G-lim cultures and decreased with increasing growth rate in both cases. Lipopolysaccharide content correlated significantly with drug uptake and sensitivity, and it appeared to determine the degree of penetration of the cell envelope by these chlorinated phenols.     

Effects of chlorhexidine gluconate on the development of spores of Bacillus subtilis

The effects of sublethal concentrations of the membrane-active agent chlorhexidine gluconate (CHG) on the growth rate and sporulation of Bacillus subtilis vegetative MB₂ cells have been investigated. CHG increased the mean generation time (Mgt) of vegetative cells in casein medium. It also affected spore development: as CHG concentrations increased, spore index (SI) values decreased and sensitivity to both toluene and heat increased.     

Adaptation and growth of Serratia marcescens in contact lens disinfectant solutions containing chlorhexidine gluconate.

Serratia marcescens (11 of 12 strains) demonstrated an ability to grow in certain chlorhexidine-based disinfecting solutions recommended for rigid gas-permeable contact lenses. For a representative strain, cells that were grown in nutrient-rich medium, washed, and inoculated into disinfecting solution went into a nonrecoverable phase within 24 h. However, after 4 days, cells that had the ability to grow in the disinfectant (doubling time, g = 5.7 h) emerged. Solutions supporting growth of S. marcescens were filter sterilized. These solutions, even after removal of the cells, showed bactericidal activity against Pseudomonas aeruginosa and a biphasic survival curve when rechallenged with S. marcescens. Adaptation to chlorhexidine by S. marcescens was not observed in solutions formulated with borate ions. For chlorhexidine-adapted cells, the MIC of chlorhexidine in saline was eightfold higher than that for unadapted cells. Cells adapted to chlorhexidine showed alterations in the proteins of the outer membrane and increased adherence to polyethylene. Cells adapted to chlorhexidine persisted or grew in several other contact lens solutions with different antimicrobial agents, including benzalkonium chloride.     

Influence of potassium chloride upon the binding and antibacterial activity of chlorhesidine diacetate and (ethoxy)5 octyl phenol (Triton X45) towards Bacillus megaterium KM-.

Chlorhexidine (0.5-0.65 microM) and Triton X45 (30-40 microM) added to exponential phase Bacillus megaterium KM- cultures was growth inhibitory. The presence of KCl (0.05-0.35 M) in the medium did not significantly affect growth rate in the absence of drug, yet reduced the growth inhibitory activity of the chlorhexidine and enhanced that of Triton X45. These effects were maximal at KCl concentrations of 0.2 M and above, when complete protection towards chlorhexidine and lysis of the cultures in the presence of Triton X45 were observed. Time-survivor curves in the presence of chlorhexidine (0.7-1.0 microM) gave LT90 values of 1.5-2.0 h in the absence of KCl, yet its inclusion (0.35 M) totally inhibited this low level bactericidal activity. Drug absorption by whole cell and isolated cell wall preparations was determined in the presence and absence of KCl (0.35 M). Chlorhexidine uptake by intact cells was reduced by approximately 50% in the presence of salt whereas that of Triton X45 increased by a similar fraction. Uptake of chlorhexidine by the cell wall fraction accounted for approximately 50% of that for the whole cells and was relatively unaffected by the presence of KCl. Conversely, absorption of Triton X45 by the cell wall fraction accounted for most of the uptake by whole cells and increased markedly in the presence of salts.     

The minimum inhibitory concentration of oral antibacterial agents against cariogenic organisms

The minimum inhibitory concentrations (MIC) of eight common dental antibacterial agents against three genera of bacteria which have been implicated in dentine caries, namely streptococci, lactobacilli and actinomycetes were investigated. The ultimate aim was to determine the most appropriate antibacterial agent which could be added to dental restorative materials for filling cavities where there was residual dentine caries. The antibacterial agents tested were chlorhexidine diacetate, chlorhexidine dihydrochloride, chlorhexidine gluconate, benzalkonium chloride, cetrimide, cetylpyridinium chloride, thymol and sodium hypochlorite. Thymol and sodium hypochlorite did not inhibit microbial growth at any of the concentrations tested. For the active antibacterial agents tested the MIC values against lactobacilli and streptococci were 0.25 microg/ml to 8.0 microg/ml and for actinomycetes 0.125 to 8.0 microg/ml. These results illustrate the wide spectrum of sensitivity of caries associated bacteria against dental antibacterial agents. From the MIC values alone, it is difficult to recommend which of the active antibacterial agents would be most effective in eliminating cariogenic organisms.     

Virion envelope content, infectivity, and neutralization sensitivity of simian immunodeficiency virus

A truncating E767stop mutation was introduced into the envelope glycoprotein of simian immunodeficiency virus (SIV) strain SIV239-M5 (moderately sensitive to antibody-mediated neutralization and lacking five sites for N-linked carbohydrate attachment) and strain SIV316 (very sensitive to neutralization, with eight amino acid changes from the neutralization-resistant parental molecular clone, SIV239). The truncating mutation increased Env content in virions, increased infectivity, and decreased sensitivity to antibody-mediated neutralization in both strains. However, the magnitude of the effect on infectivity and neutralization sensitivity differed considerably between the two strains. In the context of strain SIV239-M5, truncation increased Env content in virions approximately 10-fold and infectivity in a reporter cell assay 24-fold. The truncated SIV239-M5 was only slightly more resistant to neutralization by polyclonal monkey sera and by monoclonal antibodies than SIV239-M5 with a full-length envelope glycoprotein. In the context of strain SIV316, truncation increased infectivity a dramatic 480-fold, while envelope content in virions was increased only about 14-fold. This dramatic increase in infectivity cannot be simply explained by the increase in envelope content and is likely due to an increase in inherent infectivity, i.e., infectivity per spike, that results from truncation. The truncated SIV316 was extremely resistant to antibody-mediated neutralization. In fact, it was not neutralized by any of the antibodies tested. When increasing amounts of SIV316 envelope glycoprotein (full length) were provided in trans to SIV316, infectivity was increased and sensitivity to neutralization was decreased, but to nowhere near the degree that was obtained when truncated SIV316 envelope glycoprotein was used. Truncated forms of SIV239 and SIV239-M5 required higher levels of soluble CD4 for inhibition of infection than their nontruncated forms; truncated SIV316 did not. Our results suggest that envelope content in SIV virions, infectivity, and resistance to antibody-mediated neutralization can be increased not only by truncation of the cytoplasmic domain but also by provision of excess envelope in trans. The striking increase in infectivity that results from truncation in the context of SIV316 appears to be due principally to an increase in inherent infectivity per spike.     

Immunoelectron microscopy of Bacillus subtilis cells secreting human interferon α1 or staphylokinase

Uptake of 14C-labelled chlorhexidine diacetate (14C-CHA) by wild-type and envelope mutant strains of Escherichia coli and Pseudomonas aeruginosa was very rapid. Maximum uptake was observed within a contact time of 20 s with no additional binding on increased contact, and was concentration-dependent. In contrast to this rapid binding of 14C-CHA, bactericidal studies revealed that the lethal activity of low concentrations of unlabelled CHA was slow, although higher concentrations had a rapid effect. Comparison of a wild-type strain with its envelope mutants indicated that there was little difference in 14C-CHA uptake, in minimal inhibitory concentrations or in bactericidal activity. Azolectin was found to be an effective neutralising agent of biguanide action, but in in vitro agar tests and in reducing or removing the amount of 14C-CHA taken up by the cells.     

Shear sensitivity of hybridoma cells in batch, fed-batch, and continuous cultures.

Previously, we observed that CRL-8018 hybridoma cells were more sensitive to well-defined viscometric shear during the lag and stationary phases than during the exponential phase of batch cultures. Some potential hypotheses for explaining the increase in shear sensitivity are (1) nutrient limitations that result in a decrease in production of specific cellular components responsible for the mechanical strength of the cell, (2) nutrient limitations that lead to synchronization of the culture in a cell cycle phase that is more sensitive to shear, or (3) a link between cell growth and shear sensitivity, such that slowly growing cells are more sensitive to shear. Here, the duration of the exponential phase was increased with use of fed-batch, and the effect on shear sensitivity of the cultures was measured with a viscometric technique. Extension of exponential growth resulted in an increased period during which the cells were insensitive to shear. Additionally, the shear sensitivity of the cells was constant over a wide range of growth rates and metabolic yields in chemostat cultures. These observations suggest that as long as the cells are actively (exponentially) growing, their shear sensitivity does not depend on the growth rate or metabolic state of the cell as expressed by metabolic yields. Thus, hypothesis 3 above can be dismissed.     

Changes in the nuclear structure in the radiation-sensitive CHO mutant cell, xrs-5

The structural organization of the cell nucleus was investigated by transmission electron microscopy in the radiosensitive Chinese hamster ovary (CHO) cell mutant, xrs-5 (D0 = 45 cGy), relative to parental K1 cells (D0 = 200 cGy). In 99% of all xrs-5 cells, the outer layer of the nuclear envelope was separated from the inner layer, while 96% of K1 cells had closely apposed layers. This separation of the inner and outer layers of the nuclear envelope in xrs-5 cells was not explained by an increased susceptibility of xrs-5 cells to osmotically induced changes because (1) xrs-5 cells retained the altered nuclear periphery even when several different fixation protocols were used and (2) xrs-5 cells were not more susceptible to cell lysis as measured by trypan blue dye exclusion or by the extracellular presence of lactate dehydrogenase. The difference in the morphological organization in the nuclear periphery of xrs-5 cells correlated with the radiation sensitivity of the cells; xrs-5 cells which spontaneously reverted to a radiation sensitivity similar to that of K1 cells also reverted to a nuclear morphology similar to that of K1 cells. The inner and outer layers of the nuclear envelope were retained in nuclear scaffolds isolated from K1 and xrs-5 cells, indicating that components of the nuclear periphery are part of the nuclear scaffold. These data show that xrs-5 cells have an altered nuclear periphery which correlates with the radiation sensitivity of the cells. The separation of the layers of the nuclear envelope may represent an altered template for repair of DNA damage at the nuclear scaffold and thus may play a role in the defective repair of X-ray-induced DNA double-strand breaks in xrs-5 cells.     

Use of steroids to monitor alterations in the outer membrane of Pseudomonas aeruginosa.

Testosterone (a strongly hydrophobic steroid) and testosterone hemisuccinate (a negatively charged derivative) were used as probes to investigate alterations in the outer membrane of Pseudomonas aeruginosa. Diffusion rates of the steroids across the lipid bilayer were measured by coupling the influx of these compounds to their subsequent oxidation by an intracellular delta1-dehydrogenase enzyme. Wild-type cells of P. aeruginosa (strain PAO1) were found to be 25 times more permeable to testosterone than to testosterone hemisuccinate. The uptake of the latter compound appeared to be partially dependent on the external pH, thus suggesting a preferential diffusion of the uncharged protonated form across the cell envelope. Using various PAO mutants, we showed that the permeation of steroids was not affected by overexpression of active efflux systems but was increased up to 5.5-fold when the outer membrane contained defective lipopolysaccharides or lacked the major porin OprF. Such alterations in the hydrophobic uptake pathway were not, however, associated with an enhanced permeability of the mutants to the small hydrophilic molecule N,N,N',N'-tetramethyl-p-phenylene diamine. Thirty-six agents were also assayed for their ability to damage the cell surface of strain PAO1, using testosterone as a probe. Polymyxins, rBPI23, chlorhexidine, and dibromopropamidine demonstrated the strongest permeabilizing activities on a molar basis in the presence of 1 mM MgCl2. These amphiphilic polycations increased the transmembrane diffusion of testosterone up to 50-fold and sensitized the PAO1 cells to hydrophobic antibiotics. All together, these data indicated that the steroid uptake assay provides a direct and accurate measurement of the hydrophobic uptake pathway in P. aeruginosa.     

Transition of human breast cancer cells from an oestrogen responsive to unresponsive state

An in vitro model system is described for studying the problem of loss of steroid sensitivity in breast cancer cells. Growth of cloned oestrogen-sensitive human breast cancer cells in the long-term absence of steroid gives rise to a population of oestrogen-insensitive cells. In ZR-75-1 cells, the effect is clonal but occurs at high frequency suggesting a mechanism affecting a wide proportion of the cell population synchronously. This does not involve any reduction in oestrogen receptor number. Furthermore, there is no coordinated loss of oestrogen-sensitive molecular markers, showing that oestrogen receptors remain not only present but functional. These growth changes are not accompanied by any loss of growth inhibition by antioestrogen. Although steroid deprivation does not result in loss of oestrogen-sensitive markers, this does not hold true for other steroids. There was a reduction in progestin, androgen and glucocorticoid regulation on transfected LTRs. Loss of steroid-sensitive growth was accompanied by changes in response to exogenous growth factors and altered endogenous growth factor mRNA production. Steroid-deprived T-47-D cells acquire sensitivity to stimulation by TGFβ and have raised TGFβ₁ and TGFβ₂ mRNA levels. ZR-75-1 cells are growth inhibited by TGFβ and have reduced TGFβ₁mRNA levels. In MCF-7 cells, increased IGFII mRNA, following transfection, can result in an increased basal cell growth rate in the absence of steroid. These findings are discussed in relation to possible autocrine mechanisms in the loss of steroid sensitivity of breast cancer cells.     

Effect of chlorhexidine and benzalkonium chloride on bacterial biofilm formation

AIM: To study the effect of antiseptics on bacterial biofilm formation. METHODS AND RESULTS: Biofilm formation and planktonic growth were tested in microtiter plates in the presence of antiseptics. For Escherichia coli G1473 in the presence of chlorhexidine or benzalkonium chloride, for Klebsiella pneumoniae CF504 in the presence of chlorhexidine and for Pseudomonas aeruginosa PAO1 in the presence of benzalkonium chloride, biofilm development and planktonic growth were affected at the same concentrations of antiseptics. For PAO1 in the presence of chlorhexidine and CF504 in the presence of benzalkonium chloride, planktonic growth was significantly inhibited by a fourfold lower antiseptic concentration than biofilm development. For Staphylococcus epidermidis CIP53124 in the presence of antiseptics at the minimal inhibitory concentration (MIC), a total inhibition of biofilm formation was observed. For Staph. epidermidis exposed to chlorhexidine at 1/2, 1/4 and 1/8 MIC, or to benzalkonium chloride at 1/8, 1/16 or 1/32 MIC, biofilm formation was increased from 11.4% to 22.5% without any significant effect onto planktonic growth. CONCLUSIONS: Chlorhexidine and benzalkonium chloride inhibited biofilm formation of different bacterial species but were able to induce biofilm development for the Staph. epidermidis CIP53124 strain at sub-MICs. SIGNIFICANCE AND IMPACT OF THE STUDY: Sublethal exposure to cationic antiseptics may contribute to the persistence of staphylococci through biofilm induction.     

Quenching of the antibacterial activity of chlorhexidine and benzalkonium by Letheen broth and Letheen agar in relation to wild-type and envelope mutant strains of gram-negative bacteria

Letheen broth and Letheen agar have been investigated for their ability to act as neutralising and recovery media for wild-type and envelope mutants exposed to chlorhexidine diacetate and benzalkonium chloride. At high dilutions, untreated cells of the envelope mutant of Pseudomonas aeruginosa 799/61 were unable to produce colonies on Letheen agar. As a result of various procedures, it was concluded that dilution in Letheen broth and plating in Isosensitest agar was a suitable method for quenching cationic bactericides without harming the test strains, and that the increasing use of Gram-negative bacteria with outer membrane defects means that considerable care may be necessary in selecting media for evaluating bactericidal activity.     

Mutation that suppresses the protein export defect of the secY mutation and causes cold-sensitive growth of Escherichia coli.

A cold-sensitive mutant was isolated among temperature-resistant revertants of the secY24 mutant defective in secretion of envelope proteins across the cytoplasmic membrane at 42 degrees C. A single mutation, designated ssyA3, is responsible both for the extragenic suppression of secY and for the cold-sensitive growth. In contrast to the parental secY24 mutant, the suppressed cells do not accumulate precursors of envelope proteins at any temperatures. The cells containing the ssyA3 mutation, whether in combination with secY24 or not, show an optimal growth at 42 degrees C and a very poor growth at 30 degrees C. At the low temperature, protein synthesis is generally slowed down, probably at the step of chain elongation. The gene ssyA was mapped at a new locus between hisS and glyA on the chromosome. It is possible that the product of this gene interacts both with the protein secretion system and the protein synthesizing system.