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Biomedical subjects

J R Furr

Publications and source records attributed to J R Furr.

At least 19 recordsLinked to original sources
PubMed2004

Resistance, biguanide sorption and biguanide-induced pentose leakage during encystment of Acanthamoeba castellanii.

AIMS: This study investigates the effects of biguanides during encystment of Acanthamoeba castellanii. METHODS AND RESULTS: A non-nutrient encystment system was used to investigate the changes in the levels of sorption (uptake) of three non-cysticidal concentrations (10, 20 and 50 microg ml(-1)) of chlorhexidine diacetate (CHA) and polyhexamethylene biguanide (PHMB) as well as their effects on viability and leakage of pentose sugars during the first 36 h of encystment. Trophozoites treated with CHA or PHMB were more sensitive and generally sorbed more of each biocide than cysts. During encystment, the largest increases in resistance developed between 18 and 36 h for both biguanides with the resistance emerging to biguanide concentrations of 10 or 20 microg ml(-1) between 18 and 24 h. At 50 microg ml(-1) resistance emerged between 24 and 36 h. There was a general decrease in biocide sorption during encystment between 0-24 and 0-21 h for CHA and PHMB, respectively, at a concentration of 50 microg ml(-1). The greatest decline in biguanide-induced pentose leakage was between 0 and 12 h. CONCLUSIONS: The results suggest that during encystment some of the changes in the susceptibility to CHA or PHMB may be related to decreases in the levels of biocide sorption, which is limited by the developing cyst wall. SIGNIFICANCE AND IMPACT OF THE STUDY: During encystation, changes occur in biguanide sensitivity. The physical barrier of the cyst wall may be an important factor in limiting biocide sorption.

Acanthamoeba↗
PubMed2000

Outer membrane changes in Pseudomonas stutzeri resistant to chlorhexidine diacetate and cetylpyridinium chloride.

Changes in outer membrane proteins (OMP) and lipopolysaccharide (LPS) in cells of strains of Pseudomonas stutzeri sensitive and resistant to chlorhexidine diacetate (CHA) or cetylpyridinium chloride (CPC) have been examined. Four of five CHA-resistant strains had alterations in OMP profiles, including the expression of two additional protein bands. All the CPC-resistant strains had altered OMP profiles but the changes varied from strain to strain. Loss of the fastest-migrating bands was observed in the LPS from CHX-resistant strains. In strain JM 302R with high-level CHX resistance (minimal inhibitory concentration 100 mg/l as opposed to 2.5 mg/l in the parent-strain, JM 302), all the fast-migrating bands were lost and the strain showed 'cross-resistance' to polymyxin, gentamicin and ethylenediamine tetraacetic acid. It is however, possible that the altered LPS patterns reflect a response to alterations in other components rather than being directly associated per se with the enhanced resistance.

Anti-Infective Agents, Local↗
PubMed2000

Cytological changes in chlorhexidine-resistant isolates of Pseudomonas stutzeri.

Transmission electron microscopy (TEM), scanning electron microscopy (SEM) and energy-dispersive analysis of X-ray (EDAX) have been used to examine chlorhexidine diacetate (CHA)-sensitive and -resistant isolates of Pseudomonas stutzeri and to determine the effects of CHA on the cells. Significant differences were observed in the structure, size and elemental composition of CHA-sensitive and -resistant cells. Treatment with CHA produced considerably greater changes in CHA-sensitive cells, with widespread peeling of the outer membrane, a substantial loss of cytoplasmic electron-dense material and extensive lysis. Cells from the resistant isolates showed no blebbing of the outer membrane and no structural damage. X-ray mapping confirmed the difference in CHA uptake between CHA-sensitive and CHA-resistant cells. It is proposed that changes in the outer membrane form a major mechanism of resistance to CHA in P. stutzeri.

Chlorhexidine↗
PubMed2000

Emergence of resistance to biocides during differentiation of Acanthamoeba castellanii.

A synchronous encystment method was used to study the order of development of resistance of Acanthamoeba castellanii to a range of biocides. The emerging resistance during encystation to short-term exposure to the minimum amoebicidal concentrations of each biocide tested was recorded during the first 36 h of the differentiation process. Hydrochloric acid and moist heat were tested as possible resistance markers. Development of the acid-insoluble, proteincontaining, ectocyst wall and the cellulose endocyst wall was followed by quantification of the acid- and alkali-insoluble residues of cell samples removed from synchronous encystment cultures up to 36 h. Resistance to chemical agents (polyhexamethylene biguanide, benzalkonium chloride, propamidine isethionate, pentamidine isethionate, dibromopropamidine isethionate, hydrogen peroxide) and to moist heat was seen to develop between 14 and 24 h after trophozoites were inoculated into the encystment media. Resistance to hydrochloric acid developed between 0 and 2 h and to chlorhexidine diacetate between 24 and 36 h. Levels of acid-insoluble residues began to increase after 8 h and alkali-insoluble residues (cellulose) were detected after 16 h and coincided with the emergence of resistance to all the agents tested except hydrochloric acid. The results suggest that resistance to the biocides tested probably results largely from the physical barrier of the cyst walls rather than as a consequence of a metabolically dormant cyst.

Acanthamoeba↗
PubMed1999

Effect of permeabilizers on antibiotic sensitivity of Pseudomonas aeruginosa.

Agents which had previously been shown to act as permeabilizers against Pseudomonas aeruginosa or other Gram-negative bacteria were tested to determine whether susceptibility to various antibiotics could be increased. In the absence of a permeabilizer, Ps. aeruginosa was resistant to several hydrophobic antibiotics and vancomycin, but not to gentamicin. Tartaric and gluconic acids had weak potentiating activity, whereas ethylenediamine tetraacetic acid and citric acid were more effective permeabilizers. However, sodium polyphosphate enhanced the activity of erythromycin, fucidin, novobiocin, rifampicin and methicillin; vancomycin was unaffected and the activity of gentamicin was reduced considerably.

Anti-Bacterial Agents↗
PubMed1999

X-ray microanalysis of chlorine and phosphorus content in biguanide-treated Acanthamoeba castellanii.

Energy dispersive analysis of X-rays (EDAX) was used to study the effects of chlorhexidine diacetate (CHA) and polyhexamethylene biguanide (PHMB) on Acanthamoeba castellanii. A high variation of elements occurred in untreated individual cells and only two elements, Cl (a biocide marker) and P, were investigated. X-ray dot mapping of untreated trophozoites and cysts revealed that Cl in cells was uniformly distributed throughout the cytoplasm, whereas P was less dense in the vacuoles. X-ray dots of Cl in biocide-treated trophozoites and cysts appeared denser and evenly distributed within the cells as the biguanide concentration increased. Quantitative analysis of either CHA or PHMB within the cells using Cl as an elemental marker was unsatisfactory because of the high Cl levels in untreated cells. The apparent increases of P in some experiments with treated cells might be associated with reduced permeability, protein coagulation or aggregation of phospholipids.

Acanthamoeba↗
PubMed1999

Rapid evaluation of biocidal activity using a transposon-encoded catechol 2,3-dioxygenase from Pseudomonas putida.

Pseudomonas putida (UWC1), containing a genetically-engineered plasmid (pQM899), that encodes for the production of catechol 2,3-dioxygenase (C230), was used as a potential means of rapidly estimating bactericidal activity of chlorhexidine diacetate (CHA), phenol, cetylpyridinium chloride (CPC) and phenylmercuric nitrate (PMN). Enzyme C230 converts catechol to 2-hydroxymuconic semialdehyde (2-HMS), which is yellow in colour, via a meta cleavage pathway. Ideal conditions for production and measurement spectrophotometrically of 2-HMS were determined. However, the correlation between this method and viable plate counts was not sufficiently accurate to enable 2-HMS production to provide a sufficiently sensitive determination of biocidal activity. An alternative method, synchronous scanning fluorimetry, in which the decrease in catechol concentration was measured under standardized conditions, provided a good dose-response histogram for all the biocides tested. Although, in comparison with plate counts, there was an underestimation of the bactericidal effects of phenol an PMN, the results of this study suggest that this method has potential in determining the bactericidal efficacy of agents such as CHA and CPC.

Catechol 2,3-Dioxygenase↗
PubMed1999

Comparative responses of Pseudomonas stutzeri and Pseudomonas aeruginosa to antibacterial agents.

The sensitivity of six strains of Pseudomonas stutzeri (NCIMB 568, 10783, 11358, 11359, JM 302, JM 375) to cationic antiseptics, mercury compounds, the parabens, phenolics, EDTA and various antibiotics was compared with Pseudomonas aeruginosa NCIMB 8626. All Ps. stutzeri strains were highly sensitive to chlorhexidine diacetate, organomercurials and triclosan, but rather less so to quarternary ammonium compounds (QACs). They were also sensitive to other biocidal agents and more sensitive to many antibiotics than the strain of Ps. aeruginosa. There was little correlation between uptake of chlorhexidine diacetate or cetylpyridinium chloride by dense suspensions of organisms, leakage of intracellular constituents and loss of cell viability.

Anti-Bacterial Agents↗
PubMed1999

Development of resistance to chlorhexidine diacetate and cetylpyridinium chloride in Pseudomonas stutzeri and changes in antibiotic susceptibility.

Strains of Pseudomonas stutzeri developed stable resistance to chlorhexidine diacetate (CHA) or cetylpyridinium chloride (CPC) when exposed to gradually increasing concentrations of either antibacterial agent. Such strains showed reduced sensitivity to other non-antibiotics, including triclosan, and to some antibiotics, although this varied from strain to strain. Resistant strains were inactivated less readily by CHA or CPC and were less sensitive to sodium dodecyl sulphate. Some CHA-resistant and some CPC-resistant strains were more hydrophobic than the parent strains. Alterations in the cell envelope are likely to be responsible for non-specific changes in sensitivity to several antibacterial agents. Attempts to transfer CHA or CPC resistance by conjugation were unsuccessful. DNA from some CHA- or CPC-resistant strains could transform Ps. stutzeri strain JM 302, a histidine auxotroph, to prototrophy.

Anti-Bacterial Agents↗
PubMed1998

Acanthamoeba castellanii: growth, encystment, excystment and biocide susceptibility.

Stages in the encystment and excystment processes of Acanthamoeba castellanii have been studied. The kinetics of encystment involved measurements of the three phases (pre-encystment, cyst initiation and cyst wall synthesis). Excystment, starting from a mature cyst, involved pre-emergence, penetration outwards of the cyst wall and free trophozoite. The sensitivity to biocides of trophozoites in the exponential growth phase, pre-encystment trophozoites, mature cysts and pre-excystment cysts has been investigated. Some differences in relative sensitivity to a bisbiguanide (chlorhexidine) and a polymeric biguanide (polyhexamethylene biguanide) were observed, but mature cysts were always the most resistant cellular form.

Acanthamoeba↗
PubMed1998

Biguanide-induced changes in Acanthamoeba castellanii: an electron microscopic study.

Trophozoites and cysts of Acanthamoeba castellanii were exposed to chlorhexidine diacetate (CHA) and polyhexamethylene biguanide (PHMB); changes in cell ultrastructure and surface structure were examined by both transmission and scanning electron microscopy. PHMB caused a greater degree of structural and membrane damage; the cytoplasmic contents were severely depleted and there were clusters of densely stained precipitates on the cell surface. Concentrations of CHA greater than 100 microg ml(-1) produced shrinkage from the cyst wall. At high concentrations, PHMB induced a slight withdrawal of the cytoplasm from the wall and, unlike CHA, induced swelling of the cysts. These findings do not define the mechanisms of action of CHA and PHMB, but provide evidence that a major target site for both agents is the plasma membrane. However, additional intracellular damage undoubtedly contributes to the lethal effects. The greater resistance of cysts may be associated with reduced biguanide uptake.

Acanthamoeba↗
PubMed1998

Use of the Malthus-AT system to assess the efficacy of permeabilizing agents on the activity of antibacterial agents against Pseudomonas aeruginosa.

The Malthus-AT system provided a satisfactory method for examining the effects of permeabilizing agents on the activity of sub-inhibitory concentrations of antibacterial agents against Pseudomonas aeruginosa G48. Under this system, disodium edetate potentiated the activity of chlorhexidine diacetate (CHA), cetylpyridinium chloride, para-chlorometaxylenol and triclosan. Nitrilotriacetic acid enhanced the activity of some of the antibacterials tested, whereas sodium polyphosphate markedly reduced the efficacy of CHA.

Anti-Bacterial Agents↗
PubMed1998

Effect of permeabilizing agents on antibacterial activity against a simple Pseudomonas aeruginosa biofilm.

A simple Pseudomonas aeruginosa G48 biofilm on stainless steel discs provided a useful primary screen of potentiating effects of various permeabilizing agents on antibacterial agents. Experiments with Ps. aeruginosa suspensions could not be used to predict the effects of biocides and permeabilizers on biofilms. Although antibacterial activity against biofilms was less than demonstrated in suspension tests, potentiation by some permeabilizers was still observed.

Anti-Infective Agents, Local↗
PubMed1998

Effect of divalent cations on permeabilizer-induced lysozyme lysis of Pseudomonas aeruginosa.

The presence of divalent (Mg2+) ions greatly reduced the lysis of Pseudomonas aeruginosa strain G48 in a system at pH 7.8 or 9.0 consisting of ethylenediamine tetraacetic acid (EDTA), lysozyme and tris. Similar reductions in lysis occurred when EDTA was replaced by nitrilotriacetic acid, sodium citrate or sodium polyphosphate. The effect depended on the cation concentration. Mg2+ may replace cations removed from the outer membrane, or may effectively remove the permeabilizer from the system. The results suggest that the permeabilizing activity associated with these agents against this organism has a common basis in affecting the outer membrane.

Bacteriolysis↗
PubMed1997

Interaction of silver nitrate with readily identifiable groups: relationship to the antibacterial action of silver ions.

Microbiologically it was demonstrated that amino acids, e.g. cysteine (CySH), and other compounds, e.g. sodium thioglycollate, containing thiol groups neutralized the activity of silver nitrate against Pseudomonas aeruginosa PAO1. Amino acids with disulphide bonds were inactive, with the exception of L-cystine dimethyl ester, as were all amino acids with no sulphur groups. Iodoacetamide reacted with CySH to produce a CyS-acetamide complex that was unable to quench the activity of Ag+. Chemical analyses using cyclic voltammetry demonstrated that high coordination numbers (3.1) were obtained with thiol-containing amino acids and low numbers (0.28-0.4) with other amino acids. Both microbiologically and chemically, the results imply that interaction of Ag+ with thiol groups plays an essential role in bacterial inactivation.

Amino Acids↗
PubMed1997

Effects of biocides on Acanthamoeba castellanii as measured by flow cytometry and plaque assay.

The lethal effects of various biocides, in particular two agents (chlorhexidine diacetate and polyhexamethylene biguanide) on cysts and trophozoites of Acanthamoeba castellanii have been measured by both plaque assay and flow cytometry (FCM). Minimum amoebicidal concentrations against trophozoites were generally the same by the two methods whereas minimum cysticidal concentrations were occasionally higher by FCM. Concentrations of biguanides required to produce an equal reduction in viability were also higher by FCM than by plaque assay.

Acanthamoeba↗