In vivo standardization of cutaneous bactericidal activity of antiseptics by using monoxenic hairless mice.

This study was designed to investigate the bactericidal activities of antiseptics on the cutaneous flora of hairless mice monoxenic to Staphylococcus epidermidis, Staphylococcus aureus, or Pseudomonas aeruginosa in vivo. A standardized method for testing such antiseptics to compare their bactericidal effectiveness in humans in described. Seven antiseptics belonging to seven different chemical groups (iodine derivatives, alcohols, mercury compounds, quaternary ammonium salts, biguanides, phenols, and carbanilides) were used as recommended by the manufacturers (conditions of contact or prolonged contact time followed by washing with distilled water). Germfree hairless mice were infected with various bacterial strains by gastric intubation, producing levels of about 10(3) CFU/cm2 of skin. The antiseptic under test was placed on the right or left side of the ventral region. The contralateral side served as a control. In order to standardize the method, a number of crucial parameters were carefully controlled: the amount of antiseptic applied, the area of skin treated, the duration of treatment, and the washing procedure. Skin samples were obtained by cutaneous biopsy, which effectively removed all the bacteria along with the sample. The bacterial populations were counted before and after application of the antiseptic. Reductions of between 0.5 and 1.9 log units were obtained; these are comparable to those observed in humans. The standardization of our procedure and the use of animals with a strictly controlled flora eliminated much of the variability and sources of error inherent in human studies. This model could be of value for the study of resistant bacterial strains responsible for nosocomial infections and for investigations of damaged skin.     

Comparison of the bactericidal properties of four topical antiseptics using the resident bacterial population of the lower axillary margin

Four proprietary antiseptic creams were compared by a procedure designed to assess their bactericidal activity on human skin. The technique which employed the lower axillary margin as a test site, a 5 h contact period and bacterial sampling with alginate swabs, proved capable of demonstrating bactericidal effects and differences in product efficacy. Used in conjunction with tests in vitro , the technique can provide a useful basis upon which to compare and select antibacterial formulations for further evaluation and development.     

The cutaneous microbiology of haired and hairless mice

H arnby , D., G owland , G., H olland , K.T. & K earney , J.N. 1990. The cutaneous microbiology of haired and hairless mice. Journal of Applied Bacteriology 69 , 686–691.
The cutaneous microflora of the mid-dorsal area of hairless and haired mice was studied by processing skin biopsies. In both C3H and CBA hairless genotype animals the prevalence of colonization and the bacterial density were significantly greater than in the haired animals. The dominant bacteria were staphylococci and aerobic coryneforms. No propionibacteria were isolated. Temporal studies with C3H mice showed that from 0 to 9 days after birth the cutaneous microflora reduced and from then on the haired genotype animals maintained a low cutaneous microflora, whilst hairless genotype animals gradually lost hair from head to tail and the microflora density increased. Reciprocal skin grafting between haired and hairless animals showed that the donor skin acquired the microflora characteristics of the recipient animal after 15 d post-grafting even though the donor skin remained morphologically true to genotype.     

Maintenance of the normal flora of human skin grafts transplanted to mice

Full-thickness human cadaver skin was maintained on the dorso-lateral thoracic region of hairless mice whose immune rejection mechanism was suppressed using anti-mouse-thymocyte globulin. The bacterial profile of the pregrafted skin did not differ significantly from the normal human microflora. In contrast, the murine skin exhibited quantitative and qualitative differences from the human flora, in particular by the complete absence of Propionibacterium acnes, the dominant bacterium on sebum-rich areas of human skin. The normal microbial profile of the human grafts was maintained throughout the experimental period despite the novel environmental milieu. There was little contamination of the grafts from the normal murine flora. It was concluded that the grafted human skin would provide a realistic model for studying the ecology of human cutaneous micro-organisms.     

An evaluation of the suitability of the European suspension test to reflect in vitro activity of antiseptics against clinically significant organisms

The effectiveness of four antiseptics representing soluble phenolics (Dettol), Quaternary Ammonium Compounds (QAC) (Dettol Hospital Concentrate: DHC), mixed QAC/chlorhexidine (Hibicet Hospital Concentrate: HHC) and povidone iodine (Betadine) was assessed using the proposed phase 2 step 1 European Suspension test. The in vitro activity of the antiseptics against two of the proposed challenge strains, i.e. Staphylococcus aureus and Pseudomonas aeruginosa, was compared with that of 14 problematic clinical isolates of bacteria from a range of genera, including some multiple antibiotic resistant strains, and a clinical isolate of Candida albicans. In addition to the 5 min contact time recommended in the European test, a 1 min time was included. All four products, at their recommended use dilutions and a contact time of 5 min, achieved a Microbicidal Effect (ME) log reduction of at least 5 against the majority of organisms. Differences in activity between products were more pronounced and therefore the tests more discriminatory, when the contact time was reduced to 1 min. The clinical strains were not overtly more resistant to antiseptics than the standard test strains, suggesting that the CEN test strains mimic the antiseptic susceptibility of clinical isolates.     

Increased susceptibility to Staphylococcus aureus colonization of the skin of the NOA mouse: a potentially useful animal model for evaluating antiseptic effects.

Isolation of bacteria from wet skin lesions was attempted using Naruto Research Institute Otsuka Atrichia (NOA) mice, which develop such lesions spontaneously at a high rate. As a result, Staphylococcus aureus was demonstrated to have colonized the wet skin lesions at high density. In addition, the isolated S. aureus was found to be similar to the strain of S. aureus thought to colonize the eczematous lesions seen in humans with atopic dermatitis. Furthermore, a survey of the S. aureus colonization status of NOA mice with no wet skin lesions confirmed colonization at higher density than in HR-1 mice as control, indicating that the skin of the NOA mouse has the novel characteristic of increased susceptibility to S. aureus colonization. Thus, by using changes in S. aureus counts as an index, the NOA mouse can be expected to serve as a useful animal model for evaluating the effects of topical antiseptics. The antiseptic effects of an ointment and a lotion containing chlorhexidine gluconate were confirmed using this animal model.     

Strain differences in the induction of mono-oxygenase activity in mouse skin by topical clobetasol propionate: evidence of a role for the hr locus

The effect of the topical application of clobetasol propionate on cutaneous ethoxycoumarin O'dealkylation (EOD) has been studied in various strains of mice. Clobetasol propionate markedly increased cutaneous EOD activity in adult hairless mice only. Similar treatment of adult haired C57BL/6J mice, or adult haired DBA/2J mice had no significant effect on cutaneous EOD activity. In contrast 3 methylcholanthrene induced cutaneous EOD activity in both hairless and C57 strains to a far greater extent than in the DBA strain. EOD activity in hairless mice non-responsive to polycyclic hydrocarbons, derived by selective breeding of hairless and DBA strains was induced by clobetasol propionate to a similar extent to that observed in responsive hairless strains. Hepatic EOD activity was not induced by clobetasol propionate in any of the strains tested. Strain differences in the induction of EOD by clobetasol propionate were not related to differences in either the concentration of cytosolic glucocorticoid receptor in the skin, the dissociation constant of the cytosolic receptor, or differences in percutaneous absorption. Polycyclic hydrocarbons did not compete with triaminolone acetonide for binding to the cytosolic glucocorticoid receptor. Strain differences in the induction of EOD activity by clobetasol propionate appear therefore not to be related to strain differences in either the Ah receptor of the glucocorticoid receptor, but to be regulated by the hr locus.     

Antiseptic resistance of clinical strains of Staphylococcus aureus

To evaluate the activity of antiseptics and the sensitivity-resistance of bacteria to antiseptics, a number of characteristics has been used, including the minimum inhibiting concentration for different strains, the frequency of statistical and clinical resistance, the antiseptic activity index. A wide spread of S. aureus strains isolated from patients with hospital infections has been revealed. Differences in the resistance of bacterial strains have been established, depending on the type of the antiseptic and the ecovar of bacteria: among hospital ecovars, resistant strains occur more frequently and can resist a wider range of antibiotics. In staphylococcal hospital ecovars the occurrence and level of resistance to a number of widely used antiseptics increase with time. In connection with a wide spread of staphylococcal hospital strains resistant to antiseptics, measures on the control of the circulation of such strains should be introduced into hospitals, and the data thus obtained should be used for the periodic reevaluation of antiseptics used in medical practice and for the choice of preparations to be used for individual therapeutic and prophylactic antisepsis.     

Evaluation of selected alcoholic antiseptics activity against clinical bacterial strains

The aim of this study was to evaluate antimicrobial activity of selected alcoholic antiseptics against clinical strains, which possessed in majority a high level of drug resistance: MRSA (7), MSSA (3), E. coli: (9): strains producing ESBL (4), P. aeruginosa: (4), E. cloacae: (3), K. pneumoniae: (3). These strains were defined by MIC value, using antibiotic agar dilution method according to NCCLS. Fourteen alcoholic antiseptics were used in this study. Beside alcohol, they contained other active substances like iodine, hydrogen peroxide, chlorhexidine. Some additional agents were included for easier application, such as: gelling, moisturizing, aromatic or coloring substances. The objective of this study was also to determine the dependence of bactericidal activity on preparations (concentration). Product undiluted and diluted two and four times in water was analyzed according to prEN 12054 standard; 30 seconds and 1 minute contact time was used. The obtained data indicate that all tested undiluted antiseptics possessed bactericidal activity described by producers. However antiseptics (dilution leads to decrease and even loss of bactericidal activity. Two-times dilution of gel almost completely inactivated the product. Antimicrobial activity after 30 seconds of contact time was not affected by presence of additional agents in the tested antiseptics.     

Clinically-Relevant Cutaneous Lesions by Nitrogen Mustard: Useful Biomarkers of Vesicants Skin Injury in SKH-1 Hairless and C57BL/6 Mice

A paucity of clinically applicable biomarkers to screen therapies in laboratory is a limitation in the development of countermeasures against cutaneous injuries by chemical weapon, sulfur mustard (SM), and its analog nitrogen mustard (NM). Consequently, we assessed NM-caused progression of clinical cutaneous lesions; notably, skin injury with NM is comparable to SM. Exposure of SKH-1 hairless and C57BL/6 (haired) mice to NM (3.2 mg) for 12–120 h caused clinical sequelae of toxicity, including microblister formation, edema, erythema, altered pigmentation, wounding, xerosis and scaly dry skin. These toxic effects of NM were similar in both mouse strains, except that wounding and altered pigmentation at 12–24 h and appearance of dry skin at 24 and 72 h post-NM exposure were more pronounced in C57BL/6 compared to SKH-1 mice. Conversely, edema, erythema and microblister formation were more prominent in SKH-1 than C57BL/6 mice at 24–72 h after NM exposure. In addition, 40–60% mortality was observed following 120 h of NM exposure in the both mouse strains. Overall, these toxic effects of NM are comparable to those reported in humans and other animal species with SM, and thus represent clinically-relevant cutaneous injury endpoints in screening and optimization of therapies for skin injuries by vesicating agents.     

Efficacy of two ethanol-based skin antiseptics on the forehead at shorter application times

Background  

Recent research suggests that alcohol-based skin antiseptics exhibit their efficacy on the resident skin flora of the forehead in less than 10 minutes. That is why we have looked at the efficacy of two ethanol-based skin antiseptics applied for 10, 2.5 and 2 minutes on skin with a high density of sebaceous glands. Each experiment was performed in a reference-controlled cross-over design with at least 20 participants. Application of isopropanol (70%, v/v) for 10 minutes to the forehead served as the reference treatment. The clear (skin antiseptic A) and coloured preparations (skin antiseptic B) contain 85% ethanol (w/w). Pre-values and post-values (immediately after the application and after 30 min) were obtained by swabbing a marked area of 5 cm² for about 10 s. Swabs were vortexed in tryptic soy broth containing valid neutralizing agents. After serial dilution aliquots were spread on tryptic soy agar. Colonies were counted after incubation of plates at 36°C for 48 h. The mean log₁₀ reduction of bacteria was calculated. The Wilcoxon matched-pairs signed-ranks test was used for a comparison of treatments.     

Use of the 'ex vivo' test to study long-term bacterial survival on human skin and their sensitivity to antisepsis

AIMS: To determine bacterial survival on human skin and their sensitivity to antisepsis. METHODS AND RESULTS: An 'ex vivo' protocol which uses human skin samples placed into diffusion cells, and electron microscopy (EM), were used to study the growth of Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa inoculated onto skin samples over a 46-h incubation period at 32 degrees C. Concurrently variation in skin pH was evaluated at different time intervals during this period. In addition the antimicrobial activity of three antiseptics against the incubated micro-organisms was assessed quantitatively with the 'ex vivo' test, while their detrimental effects against bacteria were observed by EM. All three bacteria were still present in high number after 46 h inoculation on skin, although the concentration of E. coli and S. aureus were reduced by 2.74 and 1.58 log(10) reduction, respectively, over this period of time. Electron micrographs showed clear evidence of cell division and some bacteria appeared to be embedded into the skin layers. The antiseptics tested had some antibacterial activity against bacteria incubated on skin for 3 and 10 h, and EM evidence showed some morphological damages including cellular blebbing and the presence of fibrillar material around the cells. All micro-organisms had an acidifying effect on skin samples. CONCLUSIONS: Here, it was shown that bacterial pathogens can survive and grow when incubated on human skin. In addition, it is possible that they can penetrate the stratum corneum, which can provide some protection against antisepsis. SIGNIFICANCE AND IMPACT OF THE STUDY: The apparent low bactericidal activity of biocides attributed in part to bacterial protection from skin layers is particularly important to assess in order to ensure antisepsis efficacy.     

Antiseptic sensitivity of clinical strains of Pseudomonas aeruginosa

MICs, the frequency of clinical and statistic resistance and the antiseptic activity index were studied in complex on out-of-hospital and hospital ecovars of P. aeruginosa. The forms resistant to a number of antiseptics, i.e. chloramine B, chlorhexidine, decamethoxine and dioxidine whose frequency eventually increased were shown to be widely distributed. The antiseptic sensitivity spectrum was more narrow and more heterogeneous than that of other bacteria, the heterogeneity level being dependent on the antiseptic type and bacterial ecovar. The activity of pervomur, phenol, resorcin and boric acid was higher against the clinical strains of P. aeruginosa while iodopyrin, sulfacetamide sodium and dioxidine were less active. The P. aeruginosa strains had natural resistance to cetylpyridinium chloride, rokkal, ethonium, sodium laurate and laurylsulfate and rivanol. It was recommended to assay antiseptic sensitivity of agents causing purulent inflammatory infections and to control circulation of antiseptic resistant variants of bacteria in hospitals.     

Cutaneous Injury-Related Structural Changes and Their Progression following Topical Nitrogen Mustard Exposure in Hairless and Haired Mice

To identify effective therapies against sulfur mustard (SM)-induced skin injuries, various animals have been used to assess the cutaneous pathology and related histopathological changes of SM injuries. However, these efforts to establish relevant skin injury endpoints for efficacy studies have been limited mainly due to the restricted assess of SM. Therefore, we employed the SM analog nitrogen mustard (NM), a primary vesicating and bifunctional alkylating agent, to establish relevant endpoints for efficient efficacy studies. Our published studies show that NM (3.2 mg) exposure for 12–120 h in both the hairless SKH-1 and haired C57BL/6 mice caused clinical sequelae of toxicity similar to SM exposure in humans. The NM-induced cutaneous pathology-related structural changes were further analyzed in this study and quantified morphometrically (as percent length or area of epidermis or dermis) of skin sections in mice showing these lesions. H&E stained skin sections of both hairless and haired mice showed that NM (12–120 h) exposure caused epidermal histopathological effects such as increased epidermal thickness, epidermal-dermal separation, necrotic/dead epidermis, epidermal denuding, scab formation, parakeratosis (24–120 h), hyperkeratosis (12–120 h), and acanthosis with hyperplasia (72–120 h). Similar NM exposure in both mice caused dermal changes including necrosis, edema, increase in inflammatory cells, and red blood cell extravasation. These NM-induced cutaneous histopathological features are comparable to the reported lesions from SM exposure in humans and animal models. This study advocates the usefulness of these histopathological parameters observed due to NM exposure in screening and optimization of rescue therapies against NM and SM skin injuries.     

Antiseptic susceptibility and distribution of antiseptic-resistance genes in methicillin-resistant Staphylococcus aureus

We examined the antiseptic susceptibilities and distribution of antiseptic-resistance genes qacA and smr in 98 isolates of methicillin-resistant Staphylococcus aureus obtained in 1992. Seventy-one strains were resistant to antiseptics. The qacA and smr genes were detected in 10 and 20 strains, respectively. The remaining 41 strains without qacA and smr were divided into two groups that exhibited low-level (n = 22) and high-level (n = 19) resistance to acriflavin. DNA cloning and sequencing suggested that norfloxacin-resistance gene norA was responsible for the high-level resistance to acriflavin. Our results indicated that four or more antiseptic-resistance genes exist in methicillin-resistant S. aureus and that antiseptic-resistant methicillin-resistant S. aureus strains without qacA and smr are widely spread in Japan.     

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.     

Comparison of two skin antiseptics in blood transfusion. Comparison with the results of the bacteriologic control of labile blood products

The comparison of two skin antiseptics (70 degrees alcohol and 0,5% alcoholic chlorhexidine solution) was carried out by the method of in vivo impressions. The study used 45 healthy volonteers from whom a bacteriological sample was taken from both bends of the elbow, without use of an antiseptic, and after the application of one according to usual sample taking methods. The subjects were divided into two sets according to the antiseptic being tested. The results of the cultures after 24 and 48 hours are expressed in the number of germs per cm2, which thus permits us to calculate a reduction in percentage or log 10 form, and to appreciate the efficiency of the antiseptic studied. Routine bacteriological inspection of labile blood products was carried out on products picked at random every week during 18 months of exclusive use of 70 degrees alcohol by the sample taking services. The results show a comparable in vivo effectiveness of the two antiseptics on the superficial aerobic flora of the bend of the elbow, and the ease of use of 70 degrees alcohol has led to its choice. This choice is confronted with the results of the inspection of blood products: out of 1 293 inspections, only one slight contamination (cocci gram+) was found on a unit of whole blood. These data as a whole can be compared with the work of various authors.     

Antibiotic resistance plasmids and bactericidal effect of chlorhexidine on Enterobacteriaceae

The effect of plasmid RP4 on the bactericidal effect of chlorhexidine on five Enterobacteriaceae has been tested. Escherichia coli, Serratia marcescens and Proteus mirabilis strains harbouring RP4 were more susceptible than R^- strains to this antiseptic. The role of 17 other plasmids harboured in the same bacterial host ( E. coli ) on the bactericidal effect of chlorhexidine has also been examined. Six plasmids (R751, R702, R144, RP4, pME206, S-a), of which four belonged to incompatibility group P, produced an increased bactericidal effect of chlorhexidine.     

Insights into bactericidal action of surface-attached poly(vinyl-N-hexylpyridinium) chains

The surface of polyethylene slides nanocoated with silica and derivatized with long-chain poly(vinyl-N-hexylpyridinium) becomes permanently bactericidal: it kills 90–99% of (both airborne and waterborne) wild-type and antibiotic-resistant strains of the human pathogen Staphylococcus aureus. The material created was similarly lethal to strains expressing multidrug resistance pumps, the only known mechanism of resistance to cationic antiseptics.     

Comparative study on the antimicrobial effects of Hexomedine and Betadine on the human skin flora

Studies were carried out to detect the modifications, if any, on the peri-umbilical flora of six healthy volunteers after two or three daily applications of Hexomedine solution (HEX) and Betadine solution (PVI) repeated for five consecutive days. A standardized scrubbing method was used for bacterial sampling. Surviving bacteria were selected with both selective and non-selective media, and then identified by gas chromatographic fatty acid analysis. Both antiseptics were highly effective, showing both immediate and residual antimicrobial activities. The use of HEX led to a slight increase in Gram-positive cocci and a small decrease in coryneforms, but PVI produced a marked increase in Gram-positive cocci and a sharp decrease in coryneforms. The two antiseptics, however, caused no major alteration in the cutaneous microbial population. Indeed, neither the overgrowth of Gram-negative bacilli nor the emergence of resistant species was observed.