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Biomedical subjects

J Y Maillard

Publications and source records attributed to J Y Maillard.

At least 19 recordsLinked to original sources
PubMed2003

Assessment of skin viability: is it necessary to use different methodologies?

BACKGROUND/AIM: Skin is complex and may display variable structural and metabolic change 'ex vivo'. The present study aimed to follow measures of skin viability and evaluate their usefulness as markers of viability. MATERIALS AND METHODS: We evaluated the viability of skin samples fresh or after being frozen and subsequently thawed. Assessments included histopathological appearance, lactate dehydrogenase (LDH) activity, oxygen consumption and skin pH. RESULTS: Morphological investigations of fresh and frozen skin samples using light and electron microscopy showed samples with relatively well-defined epidermis and dermis. Frozen samples showed some sign of stratum corneum fragmentation, although this was not obvious. LDH activity measured in fresh samples kept at 4 degrees C was low, but it was stable up to 7 days. Fresh samples kept at 32 degrees C had a comparable LDH activity to the ones kept in the fridge up to 4 days. Frozen samples, thawed and then kept at 4 degrees C showed a stable LDH activity after 24 h of incubation. However, frozen samples incubated at 32 degrees C demonstrated a high variability in results, with up to 800 U/L of LDH activity after 5 days of incubation. Freshly excised as well as freshly thawed samples showed the highest respiration rates. Fresh and thawed samples stored for a long period of time had a significantly lower (sometimes non-existent) oxygen consumption rate. Our results also showed an increase in the oxygen consumption rate of fresh samples being incubated at 32 degrees C for 24 h. The oxygen consumption rate for all samples reached a plateau within the 15-min measurement period and even the fresh samples did not deplete all the oxygen from the medium. Skin samples ex vivo showed a significantly higher pH than human skin in vivo, and when incubated for 46 h at 32 degrees C, fresh samples had a significantly lower pH than frozen samples. All protocols were reproducible and freshly excised and freshly thawed skin samples showed the highest rates of viability. CONCLUSION: ex vivo skin shows variation of several parameters over time. It is recommended to use two or three techniques for evaluation of skin viability including at least oxygen measurement and an enzyme assay.

Cryopreservation↗
PubMed2002

Bacterial target sites for biocide action.

Although biocides have been used for a century, the number of products containing biocides has recently increased dramatically with public awareness of hygiene issues. The antimicrobial efficacy of biocides is now well documented; however, there is still a lack of understanding of their antimicrobial mechanisms of action. There is a wide range of biocides showing different levels of antimicrobial activity. It is generally accepted that, in contrast to chemotherapeutic agents, biocides have multiple target sites within the microbial cell and the overall damage to these target sites results in the bactericidal effect. Information about the antimicrobial efficacy of a biocide (i.e. the eta-value) might give some useful indications about the overall mode of action of a biocide. Bacteriostatic effects, usually achieved by a lower concentration of a biocide, might correspond to a reversible activity on the cytoplasmic membrane and/or the impairment of enzymatic activity. The bacteriostatic mechanism(s) of action of a biocide is less documented and a primary (unique?) target site within the cell might be involved. Understanding the mechanism(s) of action of a biocide has become an important issue with the emergence of bacterial resistance to biocides and the suggestion that biocide and antibiotic resistance in bacteria might be linked. There is still a lack of understanding of the mode of action of biocides, especially when used at low concentrations (i.e. minimal inhibitory concentration (MIC) or sublethal). Although this information might not be required for highly reactive biocides (e.g. alkylating and oxidizing agents) and biocides used at high concentrations, the use of biocides as preservatives or in products at sublethal concentrations, in which a bacteriostatic rather than a bactericidal activity is achieved, is driving the need to better understand microbial target sites. Understanding the mechanisms of action of biocides serves several purposes: (i) it will help to design antimicrobial formulations with an improved antimicrobial efficacy and (ii) it will ensure the prevention of the emergence of microbial resistance.

Anti-Bacterial Agents↗
PubMed2002

Cellular impermeability and uptake of biocides and antibiotics in gram-negative bacteria.

The principal targets for antibacterial agents reside at the cytoplasm and cytoplasmic membrane, damage to other structures often arising from initial events at these loci. The gram-negative bacteria offer a complex barrier system to biocides and antibiotics, regulating, and sometimes preventing, their passage to target regions. Routes of entry differ between hydrophobic and hydrophilic agents, often with a structure dependency; specialized uptake mechanisms are exploited and portage transport can occur for pro-drug antibacterials. Uptake isotherms offer insight into the sorption process and can sometimes shed light on biocide mechanisms of action. The multi-component barrier system of gram-negative bacteria offers opportunities for phenotypic resistance development where partitioning or exclusion minimizes the delivery of an antibacterial agent to the target site. Active efflux processes are recognized as increasingly relevant mechanisms for resistance, potentially offering routes to biocide:antibiotic cross-resistance. These mechanisms may be targeted directly in an attempt to compromise their role in microbial survival.

Anti-Bacterial Agents↗
PubMed2001

Possible mechanisms for the relative efficacies of ortho-phthalaldehyde and glutaraldehyde against glutaraldehyde-resistant Mycobacterium chelonae.

AIMS: This investigation compared glutaraldehyde (GTA)-sensitive and -resistant strains of Mycobacterium chelonae and examined the effects of pretreatment of GTA-sensitive and -resistant strains of Myco. chelonae with chemical agents that interfere with cell wall synthesis. METHODS AND RESULTS: When exposed to 2% (v/v) GTA at 25 degrees C, GTA-resistant strains of Myco. chelonae dried on to glass carriers were not inactivated to any significant extent. By contrast, GTA-sensitive strains of Myco. chelonae and a strain of Myco. terrae suffered a > 6 log reduction in viability in 5 min. However, ortho-phthalaldehyde (OPA; 0.5% w/v) achieved a corresponding inactivation against two GTA-resistant strains within 5-10 and 10-20 min, respectively. Electron microscopy, using a non-aldehyde fixation process and also negative staining, failed to detect any extensive changes in GTA-sensitive and -resistant cultures exposed to GTA or OPA. Thin-layer chromatography was unsuccessful in detecting differences between GTA-resistant and -sensitive strains of Myco. chelonae. However, pretreatment of GTA-resistant cells with mycobacterial cell wall synthesis inhibitors increased their subsequent susceptibility further to OPA but not to GTA. CONCLUSION: Ortho-phthalaldehyde is an effective new biocidal agent that, at its in-use concentration, is rapidly bactericidal to non-sporulating bacteria, including GTA-sensitive and -resistant mycobacteria. SIGNIFICANCE AND IMPACT OF THE STUDY: Pretreatment of GTA-resistant cells with mycobacterial cell wall synthesis inhibitors increased their subsequent susceptibility to OPA but not to GTA.

Antitubercular Agents↗
PubMed2001

Comparison of the mycobactericidal activity of ortho- phthalaldehyde, glutaraldehyde and other dialdehydes by a quantitative suspension test.

The mycobactericidal activity of various dialdehydes has been assessed by a quantitative suspension test in both 'clean' and 'dirty' conditions. Test organisms consisted of glutaraldehyde (GTA)-sensitive strains of Mycobacterium chelonae NCTC 946, M. abscessus NCTC 10882, two GTA-resistant M. chelonae strains and M. terrae NCTC 10856 (a proposed M. tuberculosis surrogate). The aldehydes tested were a new high-level disinfectant, ortho-phthalaldehyde (OPA) at 0.5% (v/v) unadjusted pH 6.5 and pH 8, GTA at 0.5% (v/v) pH 8, glyoxal at 0.5% (v/v) pH 8 and 10% (v/v) unadjusted pH 2.8, malonaldehyde sodium salt (NaMDA) at 0.5% (w/v) pH 8 and 10% (w/v) unadjusted pH 7.5 and succinaldehyde at 0.5% (v/v) pH 8. Results showed that 0.5% acidic and alkaline OPA were rapidly mycobactericidal, under both 'clean' and 'dirty' conditions, and more importantly were active against GTA-resistant strains. The washer disinfector isolates of M. chelonae were, as expected, extremely resistant to 0.5% GTA which was slowly mycobactericidal against the other strains. Glyoxal, NaMDA and succinaldehyde were ineffective against all the strains investigated. However, a high concentration of glyoxal exhibited a slow mycobactericidal activity except with M. terrae NCTC 10856, but this was not observed with NaMDA. This evaluation, using a quantitative suspension test based on a European standard, supported the claim that OPA is an effective choice as a high-level disinfectant for medical devices.

Bacteriological Techniques↗
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

A note: ortho-phthalaldehyde: proposed mechanism of action of a new antimicrobial agent.

Ortho-phthalaldehyde (OPA) is a new aromatic dialdehyde antimicrobial agent, the mechanism of action of which has been little studied. The aims of this paper are to examine what is currently known about its mechanism of action, to compare the action with that of a widely investigated aliphatic dialdehyde, glutaraldehyde (GTA), and to put forward a hypothesis that would, in the light of current knowledge, explain how OPA inactivates micro-organisms, including GTA-resistant Mycobacterium chelonae.

Disinfectants↗
PubMed2000

Development of resistance to chlorhexidine diacetate in Pseudomonas aeruginosa and the effect of a "residual" concentration.

Stable resistance in Pseudomonas aeruginosa NCIMB 10421 was obtained by step-wise exposure to gradually increasing concentrations of chlorhexidine diacetate (CHX). Repeated exposure to a proposed "residual" (sub-MIC) concentration of CHX also created stable resistance. Resistance was also developed by a single exposure to the "residual" concentration of CHX, but this was unstable. Similar experiments with Escherichia coli and CHX or cetylpyridinium chloride resulted in no significant increase in resistance. Antibiotic susceptibility profiles of the CHX-resistant P. aeruginosa cultures showed no cross-resistance, although some of the cultures were resistant to benzalkonium chloride.

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↗
PubMed1999

Ortho-phthalaldehyde: a possible alternative to glutaraldehyde for high level disinfection.

Ortho-phthalaldehyde (OPA) was tested against a range of organisms including glutaraldehyde-resistant mycobacteria, Bacillus subtilis spores and coat-defective spores. Glutaraldehyde (GTA) and peracetic acid (PAA) were tested for comparative purposes. Both suspension and carrier tests were performed using a range of concentrations and exposure times. All three biocides were very effective (> or = 5 log reduction) against Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa in suspension tests. OPA and GTA (PAA was not tested) were also very effective against Staph. aureus and Ps. aeruginosa in carrier tests. OPA showed good activity against the mycobacteria tested including the two GTA-resistant strains, but 0.5% w/v OPA was found not to be sporicidal. However, limited activity was found with higher concentrations and pH values. Coat-defective spores were more susceptible to OPA, suggesting that the coat may be responsible for this resistance. The findings of this study suggest that OPA is effective against GTA-resistant mycobacteria and that it is a viable alternative to GTA for high level disinfection.

Bacillus subtilis↗
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

Studies on the mechanisms of the antibacterial action of ortho-phthalaldehyde.

The reaction of ortho-phthalaldehyde (OPA) with amino acids and proteins was investigated as a possible mode of action. Bacterial pellets (obtained by centrifugation) changed colour after exposure to OPA. These colours were more intense at alkaline than acidic pH. Acidic and alkaline OPA reacted with primary amino acids to form coloured products. The reaction rate accelerated with increasing pH. OPA increased the optical density of bacterial cell suspensions (an indication of protein coagulation or microbial surface or other changes in the opacity of cell constituents). The inhibition of ethylenediaminetetraacetic acid- and sodium lauryl sulphate-induced lysis was not as great as for glutaraldehyde (GTA), possibly indicating less cross-linking of amines. Interactions with primary amino groups of the outer envelope or cell wall probably play a part in the action of OPA but the level of cross-linking associated with the outer membrane does not appear to be as extensive as that of GTA. The aromatic component might allow OPA to penetrate the outer layers of cells, thus helping to explain the very high activity of OPA against Gram-negative vegetative organisms even though the degree of cross-linking seems to be less than that seen with GTA. Thus, OPA reacts strongly with primary amines and stabilizes, to some extent, the outer membrane and cell walls of vegetative organisms and this probably accounts for part, but not necessarily all, of its lethal action.

Amino Acids↗
PubMed1999

Effect of biocides commonly used in the hospital environment on the transfer of antibiotic-resistance genes in Staphylococcus aureus.

The effect of sub-minimal inhibitory concentrations of biocides, commonly used in the hospital environment, on the conjugation and transduction of plasmid pWG613 was investigated in three strains of Staphylococcus aureus. The highest transfer frequency was obtained in the conjugation experiments. A low concentration of povidone-iodine was found to significantly reduce transfer frequency by 10-fold in S. aureus SAU3/13136 mating, while other biocides had no effect at low concentrations. Cetrimide (0.0001%) was found to increase significantly transduction efficiency in S. aureus RF2 when the biocide was included in the recovery media. A low concentration of chlorhexidine or povidone-iodine reduced transduction efficiency in the same recipient. This study showed that reduction in transduction efficiency was caused by the direct effect of biocides on the recipient strains rather than on the phage 80 alpha particles.

Analysis of Variance↗
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

Antimicrobial efficacy of biocides tested on skin using an ex-vivo test.

An ex-vivo test was used to evaluate the activity of antimicrobials against three microorganisms, Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus. The ex-vivo test is a carrier test using freshly excised animal skin samples maintained in viable conditions for a short period of time. Skin samples came from a veterinary practice and were excised from either dogs or cats. The antimicrobial activity of povidone iodine, chlorhexidine diacetate, cetrimide and benzalkonium chloride was also evaluated with suspension and glass-carrier tests. Generally, the activity of the antimicrobials tested was reduced when applied to the skin surface. Apart from povidone iodine (2%) against S. aureus, the biocides investigated failed to achieve a 5 log10 reduction in bacterial titre when tested with the ex-vivo method. There was no significant difference in reduction of bacterial titres after treatment with antimicrobials between the glass-carrier and the suspension tests. Furthermore, the drying process of bacterial inoculum was less detrimental on skin than on glass surfaces. This study confirmed that the activity of a biocide tested in suspension or on an inanimate surface did not reflect its activity when tested on skin. Further development of the ex-vivo test may be useful, especially for testing the antimicrobial activity of formulations with antiseptic properties.

Animals↗
PubMed1998

Resistance of Pseudomonas aeruginosa PAO1 phage F116 to sodium hypochlorite.

The development of viral resistance to sodium hypochlorite was investigated using the Pseudomonas aeruginosa bacteriophage F116 as a model system. This phage was chosen because of its structural characteristics and former investigations conducted in this laboratory. F116 was shown to be sensitive to a sodium hypochlorite concentration of 0.0075 gl-1 (available chlorine) which produced a 5 log10 reduction in titre in a suspension test. Survival bacteriophages challenged with this sodium hypochlorite concentration were isolated, propagated and challenged again with the same and higher concentrations of the biocide. It was observed that progeny virions were becoming increasingly resistant to sodium hypochlorite challenges up to a concentration of 0.0175 gl-1 of available chlorine. It was also noticed that 1-2 log10 of F116 virions from resistant phage lysates remained sensitive to the biocide. An electron microscopical investigation of F116 resistant lysates showed that the phage resistance to sodium hypochlorite was not caused by F116 particles aggregation. Furthermore, no morphological difference between the sensitive and resistant F116 particles to sodium hypochlorite was identified.

Adaptation, Biological↗
PubMed1998

Efficacy and mechanisms of action of sodium hypochlorite on Pseudomonas aeruginosa PAO1 phage F116.

The Pseudomonas aeruginosa PAO1 phage F116 was used to investigate the viricidal activity and the mechanism of action of sodium hypochlorite. The bacteriophage was inactivated with a low concentration (0.0005% available chlorine) of the biocide prepared in tap water but it was less sensitive to a sodium hypochlorite solution prepared in ultra-pure water (0.0075% available chlorine). For all the effective concentrations of sodium hypochlorite (i.e. producing at least 4 log reduction in phage titre), F116 was readily inactivated within 30 s. Electron microscopical investigations of the phage particles challenged with sodium hypochlorite showed a wide variety of deleterious effects, some of which have not been previously observed with other biocides. The wide range of structural alterations observed suggested that sodium hypochlorite has multiple target sites against F116 bacteriophage. A 30 s exposure to sodium hypochlorite (0.001% available chlorine) produced severe damage, the number and severity of which increased with a higher concentration (0.0075% available chlorine) and with a longer contact time. These observations suggested that sodium hypochlorite inactivated F116 bacteriophage by causing structural alterations to the phage head, tail and overall structure, hence possibly releasing the viral genome from damaged capsids in the surrounding media.

Antiviral Agents↗