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Recently, the procedure for surgical hand hygiene has been switching to a two-stage method and hand-rubbing method from the traditional hand-scrubbing method. Both the two-stage and hand-rubbing methods use alcohol-based hand-rubbing after hand washing. The former requires 5 min of antiseptic hand washing, and the latter 1 min of nonantiseptic hand washing. For a prolonged bactericidal effect in terms of surgical hand hygiene, chlorhexidine gluconate (CHG) has been noted due to its residual activity. However, no detailed study comparing the disinfection efficacy and prolonged effects according to different contents of CHG and the usage of alcohol-based hand-rubbing has been conducted. The glove juice method is able to evaluate disinfection efficacy and prolonged effects of the disinfectants more accurately because it can collect not only transitory bacteria but also normal inhabitants on hands. In the present study, we examined the disinfection efficacy and prolonged effects on alcohol-based hand-rubbing containing CHG by six hand-rubbing methods and three two-stage methods using the glove juice method. In both methods, 3 mL (one pump dispenser push volume) alcohol-based hand-rubbing solution containing 1% (w/v) CHG showed the highest disinfection efficacy and prolonged effects, and no significant difference was found between the hand-rubbing and two-stage methods. In the two methods of hand hygiene, the hand-rubbing method was able to save time and cost. Therefore, the data strongly suggest that the hand-rubbing method using a one pump dispenser push volume of alcohol-based hand-rubbing solution containing 1% (w/v) CHG is suitable for surgical hand hygiene.

As a preventive action plan against gastroenteritis caused by the Norovirus (NV), we studied hand hygiene effects using with three hand rubbing products, four wet wipe products, and two functional water types using Feline Calicivirus as a Norovirus surrogate. After treatment using antiseptic hand rubbing products containing chlorhexidine, quaternary ammonium, and povidone-iodine, high inactivation detected by TCID50 was observed compared to products containing povidone-iodine, although no difference was seen in viral removal measured by the amount of viral genome copies in real-time-PCR. Among wet wipes soaked in chlorhexidine, quaternary ammonium, benzoic acid and PHMB, two groups showed viral inactivation and removal. Two products were more effective for functional water, viral decrease was seen in rinsing in running electrolyzed acid water and handwashing by soap. Results underscore the importance of selection in hand washing metheds (alternative soap and also) in preventing viral gastroenteritis.

The minimum inhibitory concentration (MIC) and bactericidal activity of chlorhexidine gluconate (CHG) were determined for methicillin-susceptible Staphylococcus aureus (MSSA), methicillin-resistant S. aureus (MRSA), Escherichia coli, Serratia marcescens, Eterobacter cloacae, Pseudomonas aeruginosa and Burkholderia cepacia, isolated from patients in medical institutions all over Japan between 2000 and 2002. The following findings were obtained. 1. The MICs of CHG against MSSA, MRSA, E. coli and B. cepacia were 0.002% or less, and those against S. marcescens, E. cloacae and P. aeruginosa were 0.008% or less. 2. Rapid and strong bactericidal effects of CHG were observed against all clinical isolates of E. coli, E. cloacae and P. aeruginosa tested even at relatively low concentrations (0.02 to 0.05%). 3. Relatively high concentration or prolonged treatment time was required to achieve sufficient bactericidal effect of CHG against some isolates of S. aureus, S. marcescens and B. cepacia. These results suggest that CHG is useful antiseptic agent or disinfectant for recent clinical isolates of various bacterial pathogens. In addition, the selection of treatment concentration and treatment time for each organism and purpose was important to obtain sufficient bactericidal effect.

Cytotoxic effect and guinea pig wound cure stage, pus fabrication presence in infected wound were compared with strong acidic electrolyzed water (AcEW) and povidone-iodine solution (PVP-I), chlorhexidine (CHG) and benzalkonium chloride (BAC). It gave the following results: In a cytotoxic test, the toxicity was recognized in 0.1%-0.01% PVP-I, in 0.0002-0.0004% CHG, in 10-0.1 micrograms/ml BAC, but there was no toxicity in AcEW. By a guinea pig wound cure process, no significance was recognized between each pharmaceutical agent in epidermal cell migration, but by an inflammation locus area, the significance was considerable in comparison with no-treatment. The pyopoiesis of P. aeruginosa infected wound was recognized in a ratio of 38.2% physiological saline, 27.3% CHG, 20.6% PVP-I and 12.1% AcEW. When pollution locus includes an infection image of bacteria, while draining AcEW instead of physiological saline, disinfection, indication was expected, and, as for the disorder in cure stage. I do not agree with that mentioned above. As for AcEW, availability by organism use was recognized for the cytotoxic effect of antiseptic instead of action of acceleration for wound cure.

Effect of chlorhexidine gluconate (CHG) on 98 clinical isolates of Serratia marcescens, previously O-serotyped, in The Showa University Fujigaoka Hospital (1989. 1-1991. 3) was investigated. The serotype O3 group (28 strains) was more frequently isolated than any other groups and more resistant to 28 different antimicrobial agents tested. 18 (50%) of 36 strains (MIC of CHG > or = 100 micrograms/ml) were serotype O3 and MIC-distribution of CHG for total O3 strains was more resistant than that for the other serotype strains. Bactericidal effect of CHG on 18 O3 strains (MIC of CHG > or = 100 micrograms/ml) was significantly lower than on the other 10 strains (MIC of CHG < or = 50 micrograms/ml). Although 19 (68%) of total O3 strains were resistant to cefotaxime and the other drugs, MIC-distribution of CHG for these strains was not associated with the antimicrobial resistance tested. The above results, at least, suggest that the serotype O3 group is suited for residing in hospitalized patients more than any other serotype groups.

A study was made on the MIC distributions of chlorhexidine and benzalkonium chloride against clinically isolated 178 strains of Pseudomonas aeruginosa to find out the existence of strains resistant to those disinfectants and also on the in vitro induction of resistance to both drugs. The MIC of chlorhexidine gluconate was found to be distributed from 78 to 625 micrograms/ml with a single peak at 312 micrograms/ml. All 178 strains of clinical isolates were sensitive to chlorhexidine and none could be induced to become chlorhexidine resistant in vitro, suggesting that P. aeruginosa can not easily acquire chlorhexidine resistance. On the other hand, the MIC of benzalkonium chloride was distributed in two peaks; one peak was benzalkonium sensitive at 625 micrograms/ml (150 strains/178 strains: 84.3%) and the another peak was benzalkonium resistant at 5,000 micrograms/ml (28 strains/178 strains; 15.7%). Six (4.0%) of the 150 benzalkonium sensitive strains acquired benzalkonium resistance by in vitro induction of resistance; the MIC of 5 strains increased from 625 micrograms/ml to 2,500 micrograms/ml and that of the residual 1 strain increased from 312 micrograms/ml to 1,250 micrograms/ml. However, no change of MIC was observed in 28 benzalkonium-resistant strains of clinically isolated P. aeruginosa by in vitro resistance induction. Strains with MIC more than 5,000 micrograms/ml could not be obtained at all. The results suggest that the benzalkonium resistance can be introduced in P. aeruginosa whereas the resistance-acquiring rate is low. These results suggest that chlorhexidine gluconate is the first choice for prevention of Pseudomonas infection in the hospital and benzalkonium is also useful in 0.5% solution is used.

A study was made on the relation between in vitro bactericidal activity against Staphylococcus epidermidis which is the main bacterial flora of skin and the disinfecting activity of hand-washing with povidone-iodine, sodium hydrochloride, chloramin-T, chlorhexidine gluconate and benzalkonium-chloride. Although the bactericidal activity of povidone-iodine and sodium hydrochloride was significantly high as compared with those of the other three disinfectants, both povidone-iodine and sodium hydrochloride showed no or a little hand-washing effects. In vitro the bactericidal activity of both povidone-iodine and sodium chloride were significantly retarded by the addition of peptone in the reaction mixture. On the other hand, chloramin-T, chlorhexidine gluconate and benzalkonium chloride showed a high disinfecting activity in hand washing in spite of low in vitro bactericidal activity. Moreover, the bactericidal activity of these three disinfectants were not suppressed by the addition of peptone in the reaction mixture. These results strongly suggested that hand washing effect of disinfectant is affected by the protein on the hand and that hand-washing activity of disinfectant should be determined by in vitro bactericidal activity in the presence of peptone.

Highly methicillin-resistant Staphylococcus aureus (H-MRSA, MIC greater than 100 micrograms/ml) was prevalent from 1986 in our institution. The failure of povidone-iodine to reduce the prevalence of MRSA led us to choose chlorhexidine-ethanol solution as an antiseptic, and then the isolation frequency of H-MRSA decreased significantly in 1988. When H-MRSA began to increase again recently, we studied the resistance to antiseptics of MRSA in order to investigate the cause of this re-increase. Common antiseptics were tested against 45 strains of H-MRSA and 22 strains of methicillin sensitive S. aureus (MSSA, MIC less than 12.5 micrograms/ml). Dilute preparations (1:100) of povidone-iodine and chlorhexidine-ethanol solution were more effective on H-MRSA than the other antiseptics. Though there was no significant difference between H-MRSA and MSSA in their sensitivity to povidone-iodine, the killing of H-MRSA strains was more delayed than the killing of MSSA strains in chlorhexidine. Even after a 120-second exposure, 13.3% of H-MRSA strains were resistant to chlorhexidine (more than 1000 colonies were recovered). These highly chlorhexidine-resistant strains have been isolated since 1987 when we chose chlorhexidine-ethanol solution as the antiseptic in our institution. Therefore we suspect that the acquirement of resistance to antiseptics by H-MRSA caused the re-increase of this strain.

The mechanisms of resistance of Serratia marcescens and Pseudomonas cepacia to chlorhexidine were studied. Leakage of the cellular component such as protein was observed in a chlorhexidine sensitive strain (S1) of S. marcescens when S1 was treated with chlorhexidine at 40 micrograms/ml concentration, while this phenomenon was not observed in a resistant strain (R1). The following observations were made concerning about cell surface structure in the chlorhexidine sensitive and resistant strains of S. marcescens by chemical analyses of membrane components and electron microscopical studies of the thin sections of the cells. (1) When the S1 strains was treated with chlorhexidine, the outer membrane of the cells formed a wrinkled surface with irregular blebs, and some of which broke out to form various sizes of granules. The R1 strain did not undergo such morphological changes under the same conditions used in the sensitive strain. (2) A prominent protein with apparent molecular weight of 45 K was found exclusively in the R1 strain of S. marcescens as major outer membrane protein, while it was not found in S1 strain. (3) There were no differences in the composition of phospholipids and the amount of 3-hydroxytetradecanoic acid between S1 and R1 strains of S. marcescens. In Pseudomonas cepacia PCJ1 which is resistant to chlorhexidine, 50 K protein was also observed as a major protein of outer membrane of the cells. In contrast to the strain, a mutant of the strain, #102 which was obtained from PCJ1 strain by the treatment with methanesulfonic acid ethylester, did not possess the 50 K protein in its outer membrane. These data suggested that outer membrane components of bacteria were related importantly in resistant mechanism.

The purpose of the present investigation was to determine bacterial contamination of chlorhexidine coated and uncoated (normal) nylon filaments of toothbrushes. Ten healthy subjects were employed in this study and the test toothbrush of four lines and thirty-eight tufts were used twice a day. Test periods were 1, 8, and 20 days for each filaments of toothbrush and after they brushed the used toothbrush was kept at the constant condition (20 degrees C, 65% RH). After each test period, those toothbrushes were immediately collected and kept at the same condition for drying. After 0, 3, 6, 9, and 24 hours of drying, two tufts of filaments were pulled out from the toothbrush and cutted into two parts (end- and root-side of tufts) by sterile scissors. Each cutted part of filaments was washed with PBS and the aliquots was incubated on Brucella blood agar plate at 37 degrees C for 48 hours. The number of bacteria attached to filaments was enumerated. The results were as follows: 1. The number of bacteria attached to both end- and root-sides of chlorhexidine coated filaments decreased with the time of drying. 2. At the root-side of the normal filaments, the number of contaminated bacteria increased with the test periods. 3. The isolated bacteria from tested toothbrushes were mainly gram positive at shorter period, however, gram negative rods were also found at longer period. 4. The anti-bacterial activity of the end-side of chlorhexidine coated filaments diminished at eight days of the use, however, their activity at the root-side still remained even at twenty days. From these results, it was clear that the bacterial contamination of toothbrush was affected by several factors such as the condition of drying, the site of tufts, the using period of toothbrush and so on. To prevent this problem, it is important to keep it at good storage condition and to change it periodically. The developed chlorhexidine coated filaments of the toothbrush also indicates to be one of the useful way for prevention of bacterial contamination of toothbrush.

Antibacterial action of various denture base resins on thirteen species of bacteria were studied in vitro. Antibacterial effect of 5% tannin-fluoride preparation, 5% tannic acid and 5% chlorhexidine added to resins on these thirteen bacterial species were also investigated using heat-curing denture base resins. Fresh microwave-curing resin and pour-type resin each showed an antibacterial action on one bacterial strain. Fresh self-curing resins had antibacterial actions on several bacterial strains. However, after storage in water at 37 degrees C for one week, antibacterial action of microwave-curing and pour-type resin were diminished and self-curing resins partially lost their antibacterial actions. Denture base resin containing tannin-fluoride preparation or tannic acid showed an antibacterial effect on one bacterial strain. On the other hand, denture base resin containing chlorhexidine had an antibacterial action on eleven bacterial strains. However, color and mechanical properties of the drug-containing resins were not satisfactory.