Effect of Antimicrobial Soap Containing Chlorhexidine on the

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1 APPLIED AND ENVIRONMENTAL MICROBIOLOGY, June 1976, p Copyright 1976 American Society for Microbiology Vol. 31, No. 6 Printed in U.S.A. Effect of Antimicrobial Soap Containing Chlorhexidine on the Microbial Flora of Skin RAZA ALY* AND HOWARD I. MAIBACH Department of Dermatology, University of California, San Francisco, California Received for publication 6 September 1975 The qualitative and semiquantitative changes in the aerobic microbial flora of normal skin with the prolonged use of a chlorhexidine scrub (6 months) were investigated. More samples in the chlorhexidine scrub group had gram-negative bacilli in their axilla (63 of 96, 66%) and groin (36 of 96, 38%) than the controls (32 of 66, 49%, for axilla and 7 of 66, 11%, for groin; P = 0.01). Klebsiella and Enterobacter were the predominant organisms in the control and chlorhexidine groups, respectively. The chlorhexidine scrub produced a reduction in the total aerobic counts in the axilla, groin, and between the toes and the fingers. Fewer samples from the chlorhexidine-treated areas revealed the presence of lipophilic diphtheroids than did the controls. Lipophilic diphtheroids were also reduced quantitatively in the groin and axilla with chlorhexidine treatment. No consistent pattern for the other major groups of bacteria was noted between the treatments. The normal flora of the skin, composed primarily of gram-positive cocci and diphtheroids, may represent a selective barrier against proliferation of potentially pathogenic organisms (13). In some individuals, small numbers of gram-negative organisms or yeasts may also comprise this normal flora, but their proliferation may be influenced by the normal ecological balance. Recent reports suggest that repeated use of antimicrobial-containing preparations may suppress the protective gram-positive population, resulting in a potentially harmful shift toward gram-negative colonization (7, 8, 11). The primary reason for this occurrence is the selectivity demonstrated by most currently popular germicides for gram-positive organisms (10, 16). The antimicrobial agent chlorhexidine has been available for use in skin antisepsis in England and Europe for 20 years. Besides being active against gram-positive organisms, chlorhexidine also displays activity against gramnegative organisms and yeasts (4, 6). This study was designed to measure effects on cutaneous microbial ecology during the long-term use of a nonionic detergent solution containing 4% chlorhexidine gluconate (Hibiclens). MATERIALS AND METHODS One-hundred and sixty healthy male volunteers were initially screened for the presence of cutaneous gram-negative rods. Subjects carrying gram-negative organisms in at least one of the four sample areas (axilla, groin, hand, or toes) were examined further. Gram-negative carriers were chosen for treatment to maximize the possibility that chlorhexidine would suppress gram-positives and allow increased gram-negative growth. The subjects selected for this study used nonmedicated soap (Ivory) for the 2-month period of screening. A total of 40 subjects was selected. On two subsequent occasions before treatment, cultures were taken from the four sample areas of each subject. Treatment. Following the base line determinations (before the treatments), 15 subjects were randomly assigned to the control group (nonmedicated) and 25 subjects were assigned to the test group (chlorhexidine). All 40 subjects bathed daily with the assigned treatment. In the chlorhexidine group, each subject washed his body (except the head) and hands for 3 min using 15 ml of chlorhexidine; after a thorough rinse, the procedure was repeated. Subjects in the control group followed the same procedure using the nonmedicated soap. All subjects were issued a perfumed deodorant not containing an antimicrobial agent and were cautioned to avoid contact with any materials (topical or systemic) that would alter the normal skin flora. Sampling of bacteria. Samples were taken at least 12 h after washing. For the finger webs and toe webs, the microorganisms were removed by rubbing moist cotton swabs over the surfaces eight times (1- by 4-cm area). For the groin and axilla, 1 ml of wash solution (nutrient broth, 10% Tween 80, and 3% Axolectin to neutralize the carry-over antimicrobial effect of chlorhexidine) was pipetted into a sterile aluminum cylinder (6.15-cm2 area of skin) while it was pressed firmly against the skin with one hand. The skin was rubbed gently with a Teflon "policeman" (a scrubbing device) for 1 min. The suspension was aspirated and serially diluted (1:100 to 1:10,000) 931

2 932 ALY AND MAIBACH with nutrient broth containing 10% Tween 80. Samples (0.1 ml) of undiluted washings were spread on eosin methylene blue, Sabouraud glucose agar, crystal violet, and pseudomonas agar. Media. Appropriate dilutions were plated within 10 min. Sheep blood agar, Trypticase soy agar with Tween 80, staph no. 110 media, eosin methylene blue media (BBL), crystal violet agar (Difco), pseudomonas agar (Difco), and Sabouraud glucose agar containing antibiotics (penicillin, 20 U/ml, and streptomycin, 40 mg/ml) were used. Sabouraud agar plates were used to estimate the total counts of Candida albicans, whereas Trypticase soy agar with Tween 80 was used for lipophilic diphtheroids. Lipophilic diphtheroids grow poorly on blood agar plates but luxuriantly in the presence of Tween 80 (15). The counts for lipophilic diphtheroids on Trypticase soy agar-tween 80 did correspond to the count on the blood plates. Plates were incubated at 37 C for 48 h. Sabouraud agar plates were incubated at room temperature. Samples from different types of colonies were picked for identification of different groups of bacteria. Gram strains were performed on selected colonies. Sheep blood agar plates were used to estimate the total count of bacteria. The total counts for C. albicans were estimated on Sabouraud agar plates and added to the total bacterial counts. Organisms were classified according to their response to biochemical tests and by their growth and/or morphology on selective or differential media. Colonial pigment, catalase production, coagulase production, oxidative or fermentative metabolism of glucose production of spores, and motility were used to identify the organisms (9). RESULTS Comparison of the regional flora in subjects using nonmedicated soap and chlorhexidine scrub: qualitative. The bacterial flora of differ- TABLE 1. APPL. ENVIRON. MICROBIOL. ent skin areas of the subjects who used a surgical scrub formulation and nonmedicated soap for 6 months is compared in Table 1. Averages of six monthly trials of the 11 control and 16 test subjects completing the study were tabulated. The occurrence of gram-negative organisms in the axilla (63 of 96 samples, 66%) and groin (36 of 96, 38%) of test subjects was higher than that in the controls (32 of 66, 49%, axilla; and 7 of 66, 11%, groin). This difference between the treatments was statistically significant (P = 0.01). More samples from the groin and toes in the chlorhexidine group carried Staphylococcus aureus than samples from the controls; no significant overall treatment differences for staphylococci were noted for the four locations. Isolation of lipophilic diphtheroids was lower in all four skin areas of chlorhexidine groups (9 versus 12%, hand; 37 versus 58%, axilla; 32 versus 55%, groin; and 30 versus 47%, toes). No consistent differences in the incidence of other diphtheroids were noted between the two treatments. Eight of the 96 (7%) samples in the chlorhexidine group revealed the presence of C. albicans in the groin; none of the controls showed the presence of C. albicans. In contrast, the toes in the control group had a higher incidence of C. albicans (11 of 66 samples, 17%) than did those in the test subjects (3 of 96, 3%). Comparison of gram-negative rods: quantitative estimation. The average counts for the base line study and during the last 2 months of treatments (chlorhexidine and nonmedicated soap) were compared (Fig. 1). Axilla. The gram-negative counts decreased Occurrence of microbial flora (percent) on the skin of subjects using chlorhexidine and nonmedicated soap" Gram- au- Gram Staphy- Micro- Lipo- Nonli- Gram- Samples' negative S. positive philic pophilic Strepto- negative C. albirods reus rods lococci cocci diphthe- diphthe- cocci cocci cans roids roids Fingerweb Control subjects Test subjects Axilla Control subjects Test subjects Groin Control subjects Test subjects Toeweb Control subjects Test subjects " Averages of six monthly trials of 11 controls and 16 test subjects completing the study are included. bcontrols, 66 samples; test subjects, 96 samples.

3 VOL. 31, 1976 from base line counts when subjects changed to chlorhexidine or nonmedicated soap. The reduction was noted in both; the decrease was slightly greater in test subjects than in the controls. This difference was not statistically significant. Groin. In contrast to the axilla, gram-negative rods increased when the subject changed to the chlorhexidine scrub; in the controls the gram-negative rods were not detected after 4 months of treatment (P = ). Thus, the controls demonstrated fewer gram-negative rods in the axilla and groins than did the chlorhexidine-treated subjects. Because there were fewer numbers and only sporadic appearance of gram-negative rods, a comparison between the fingers and the toe webs was not made. Comparison of gram-negative rods: qualitative. The occurrence of gram-negative rods in subjects using nonmedicated soap and the chlorhexidine scrub was compared (Table 2). Klebsiella were the prodominant organisms in the controls (51.3%), followed by Proteus (18.9%) and Enterobacter (16.2%). In the chlorhexidine group Enterobacter were the predominant bacteria (48.8%), followed by Klebsiella (24.4%) and Proteus (13.9%). Comparison of treatments among two major groups of organisms: quantitative. The density of staphylococci and lipophilic diphtheroids in the axilla and groin during the last 2 months of treatments was compared. The other test sites and bacteria were not included for ~~~~~~~~~ x t AXILLA i IGROIN - FIG. 1. Comparison of gram-negative rods: the average counts for the base line study and for the last 2 months of treatments (chlorhexidine and nonmedicated soap were compared). Subjects using nonmedicated soap served as controls. Counts are expressed per square centimeter. TABLE 2. a zu ANTIBACTERIAL SOAP AND SKIN FLORA 933 comparison due to their inconsistent trends. (i) Lipophilic diphtheroids. The axilla counts were lower in the chlorhexidine-treated group than in the nonmedicated soap-treated group (2.6 x 10" versus 4.7 x 1Oi/cm2, P = ). Counts associated with chlorhexidine were also lower in the groin, when compared with those in the controls (5.91 x 105 versus 2.25 x 106/cm2), and this difference in counts was not statistically significant. (ii) Staphylococci. Unlike lipophilic diphtheroids, staphylococci counts in the axilla were higher in the chlorhexidine-treated group than in the control group (6.7 x 105 versus 3.5 x 105/cm2). The counts in the groin were lower in the chlorhexidine-treated group than in the controls exposed to nonmedicated soap (2.8 x 105 versus 5.51 x 105/cm2; P = ). Total microbial counts in the chlorhexidine versus nonmedicated soap treatments. Bacterial counts over a 6-month period were collected from each site of the skin from subjects using medicated or nonmedicated products. The total aerobic bacterial counts (average of six monthly counts) of the two groups are shown in Fig. 2. Lower total microbial counts were noted in all of the four areas in subjects using chlorhexidine than in the subjects using the non-antibacterial soap. The decrease in density when compared So ,5. I 1+N I AXLLA P = '- 'I) 'V~ l l5 I"I\"I GROIN TOE WEB P =O.0056 P = o FINGER WEB P = FIG. 2. Total microbial counts in the chlorhexidine and non-antibacterial soap treatments. Bacterial counts over a 6-month period were collected from each site of the skin and averaged. Symbols: solid bar, controls; striped bar, chlorhexidine treatment. Counts are expressed per square centimeter. Occurrence ofgram-negative genera (percent) on the skin" of subjects using chlorhexidine and nonmedicated soap Treatment Enterobacter Proteus Klebsiella Escherichia Providen- Serratua Arizona cia Controls Chlorhexidine I Axilla, groin, toeweb, and finger webs. '45--l

4 934 ALY AND MAIBACH with controls was: 87% between the finger webs, 56% in the groin, 49% in the axilla, and 19% in the toe webs. DISCUSSION The suppression of cutaneous microflora by washing the skin with surface-active agents containing antimicrobial substances is a complex process involving numerous interactions (1, 2, 5, 12). The normal flora of the skin differs greatly in regard to sensitivity to a given antibacterial agent. Long-term use of soap containing bacteriostatic agents may lead to the detrimental overgrowth of a particular bacterial species that otherwise would be unable to survive on the skin. Regional flora in the subjects using the chlorhexidine scrub demonstrated a higher incidence of gram-negative organisms in the axilla (66%) and groin (38%) than did that in the control subjects (49% axilla and 11% groin). This small increase in the incidence of gramnegative organisms must be viewed in perspective. In other situations, when the resident microbial flora was drastically reduced by systemic therapy, significant overgrowth of gramnegative organisms has followed (3, 9, 11). The counts of gram-negative organisms were examined in the groin and were increased from 7.9 x 10' in the base line determination to 7.4 x 102 with chlorhexidine treatment (average counts of last 2 months of chlorhexidine treatment). In the control group the counts decreased from 8.8 x 10' to nondetectable numbers during the last 3 months of treatment. This increase of gram-negative organisms associated with chlorhexidine treatment was not noted in the axilla. On the other hand, in the axilla, gram-negative rods were reduced both in the chlorhexidine and control groups. This reduction was slightly greater in the test subjects than in the controls. These data suggest that more skin sites are needed when comparisons of less potent antimicrobial agents are made. No harmful effects due to the exaggerated use of chlorhexidine over a period of 6 months were noted. It seems that chlorhexidine treatment did lower the total microbial counts in all the test sites. Among the normal flora, lipophilic diphtheroids were the most sensitive bacteria to chlorhexidine treatment and were reduced in density with chlorhexidine treatment; staphylococci were reduced in the groin but not in the axilla. We do not have an explanation as to why contrasting results were obtained in two different regional areas with the same treatment. No specific selection of bacterial species with prolonged use of chlorhexidine was noted. APPL. ENVIRON. MICROBIOL. There are no similar published studies available for comparison to allow us to gain broader perspective in interpretation as to clinical relevance. A surgical scrub is not intended for this exaggerated usage (daily application to the whole body for 6 months). General experience suggests that long-term administration of surgical scrub to the hands only does not lead to cutaneous bacterial or fungal disease. Most organisms were not completely eradicated by topical chlorhexidine treatment. The most striking changes were in the groin (perhaps because of higher relative humidity). In the control subjects, levels of gram-negative orgamisms were undetectable at the end of the study, whereas in the treated subjects they had increased. The increase in incidence was more impressive than the increase in numbers of organisms in a given subject. Perhaps several-fold higher numbers of gram-negative organisms are required to produce clinical disease in human skin. The major anatomic sites for gram-negative skin infection in humans are the face, neck (gram-negative folliculitis), and foot (10, 16). There are monthly variations in the density of bacteria, and this fact emphasizes the importance of appropriate controls (13; unpublished data). If a nonmedicated control were not also examined, the changes noted over time might incorrectly be ascribed to the antimicrobial agent rather than to the apparently complex changes noted in our control group. The alterations in cutaneous flora seen in both groups are not assumed to represent hazard, at least in terms of our present knowledge. Much additional information on factors controlling skin flora and antimicrobial agents is needed to help explain the data shown here. ACKNOWLEDGMENTS We wish to express our thanks to Walter Lindsey and Charlene Shirley for technical assistance and to Eugene Prout of the California Medical Facility, State of California, Department of Corrections, Vacaville, Calif., for their cooperation in this investigation. LITERATURE CITED 1. Aly, R., H. I. Maibach, R. Rahman, H. Shinefield, and A. Mandel Correlation of human in vivo and in vitro cutaneous antimicrobial factors. J. Invest. Dis. 13: Aly, R., H. I. Maibach, and H. Shinefield Survival of pathogenic organisms on human skin. J. Invest. Dermatol. 58: Aly, R., H. I. Maibach, W. G. Strauss, and H. R. Shinefield Effect of systemic antibiotic on nasal bacterial ecology in man. Appl. Microbiol. 20: Beeuwkes, H The use of chlorhexidine. Antonie Van Leeuwenhoek J. Microbiol. Serol. 24: Burtenshaw, J. J The mechanism of disinfection of the human skin and its appendages. J. Hyg. 42: Davies, G. E., J. Francis, A. R. Martin, F. L. Rose, and

5 VOL. 31, 1976 G. Swain :6-DI-4'-chlorphenyldiguanidohexane ("Hibitane"). Laboratory investigation of a new antibacterial agent of high potency. Br. J. Pharmacol. 9: Ehrenkranz, N. J., D. Taplin, and P. Butt Antibiotic-resistant bacteria on the nose and skin: colonization and cross-infection, p Antimicrob. Agents Chemother Forfar, J. O., J. C. Gould, and A. F. MacCabe Effect of hexachlorophene on incidence of staphylococcal and gram-negative infection in the newborn. Lancet ii: Isenberg, H. D., J. A. Washington II, A. Balows, and A. C. Sonnenwirth Collection, handling, and processing of specimens, p In E. H. Lennette, E. H. Spaulding, and J. P. Truant (ed.), Manual of clinical microbiology, 2nd ed. American Society for Microbiology, Washington, D.C. 10. Leyden, J. J., R. R. Marples, 0. H. Mills, and A. M. Kligman Gram-negative folliculitis-a complication of antibiotic therapy in acne vulgaris. Br. J. ANTIBACTERIAL SOAP AND SKIN FLORA 935 Dermatol. 88: Light, I. J., J. M. Sutherland, M. L. Cochran, and J. Sutorius Ecological relation between Staphylococcus aureus and Pseudomonas in nursery population. N. Engl. J. Med. 278: Marples, R. R., and P. Williamson Effect of systemic dimetrylchlorotetracycline on human cutaneous microflora. Appl. Microbiol. 18: Noble, W. C., and D. A. Somerville Microbiology of human skin, p W. B. Saunders, Philadephia. 14. Rebell, G., P. M. Pillsbury, D. Phalle, and D. Ginsberg Factors affecting the rapid disappearance of bacteria placed on the normal skin. J. Invest. Dermatol. 14: Smith, R. E Characterization of human cutaneous lipophilic diphtheroids. J. Gen. Microbiol. 55: Taplin, D., N. Zaias, and G. Rebell Environmental influences on the microbiology of the skin. Arch. Environ. Health 11: Downloaded from on September 25, 2018 by guest