Hammer et al. TABLE 3 Summary of E.coli MICs of antibiotics (ug/ml)alone,antibiotics with tea tree oil (0.062%)or terpinen-4-ol (0.031%),and tea tree oil or terpinen-4-ol without antibiotics,determined by serial subculture MIC on day: Agent(no.of isolates) and parameter Treatment 1 2 3 5 6 CIP (12) Median Alone 0.008 0.016 0.016 0.03 0.03 0.12 With TTO 0.008 0.016 0.03 0.06 0.06 0.12 With T4ol 0.008 0.016 0.06 0.06 0.12 0.12 GM Alone 0.010 0.025 0.034 0.070 0.070 0.140 With TTO 0.002 0.014 0.029 0.045 0.064 0.122 With T4ol 0.009 0.018 0.048 0.078 0.100 0.147 KAN (11) ownloaded Median Alone 32 4 64 128 With TTO ￥ 16 32 3 32 4 +With T4ol Y 16 3 32 64 GM Alone 6.7 45.3 20.2 60.4 50.8 107.6 from With TTO 麷 12.7 33.9 38.1 42.7 47.9 With T4ol 151 28.5 33.9 32.0 53.8 AMP (10) http://aac. Median Alone 8 16 With TTO 6 16 6 With T4ol 2 16 GM Alone 2.1 4.9 8.0 10.6 11.3 16.0 asm.or With TTO 2.1 4.6 8.6 9.8 12.1 12.1 口 With T4ol 2.1 4.9 11.3 11.3 17.1 243 Tea tree oil (18) Median Alone 0.5 1 1 1 GM Alone 0.65 0.73 0.96 1.00 1.12 0.96 Terpinen-4-ol (18) Median Alone 0.12 0.25 0.25 0.25 0.25 0.25 吕 GM Alone 0.13 0.17 0.22 0.29 0.25 0.26 GM,geometric means.Boldface type indicates that the MIC is more than double the baseline (day 1)value.Single underlining indicates that values differed significantly from antibiotic alone on that day.Double underlining indicates significant differences between tea tree oil and terpinen-4ol treatments on that day. SHA Z tistical analysis of MICs obtained on each day under the three grow consistently in concentrations greater than 0.1%terpinen- different conditions (antibiotic alone,with tea tree oil,or with 4-ol after 18 to 20 passages,demonstrating that resistance to terpinen-4-ol)demonstrated significant differences for cipro- terpinen-4ol could not be induced in vitro (Table 4).Similarly,S. floxacin on days 1(P<0.0001)and 2 (P=0.02),for vancomycin epidermidis ATCC 12228 would not grow at concentrations above on days 2(P<0.0001),4(P=0.0017),and 6(P<0.0001),and 0.2%terpinen-4-ol,and S.aureus ATCC 25923 would not grow for mupirocin on day 2(P 0.0082). above 0.1%tea tree oil.Serial passage with terpinen-4-ol resulted For E.coli,increases in the MIC of more than two doubling in few changes in antimicrobial susceptibility(Table 1).Changes dilutions occurred for all three antibiotics alone on days 2 to 3 in MICs of two or more dilutions were evident for ciprofloxacin, (Table 3).Increases in median MICs from days 1 to 6 for antibiotic gentamicin,tetracycline,and benzalkonium chloride only.How- alone were 16-fold for ciprofloxacin and kanamycin and 8-fold for ever,with the exception of benzalkonium chloride and S.aureus ampicillin.Similarly to S.aureus,the presence of TTO or ATCC 25923,differences were not observed consistently for every terpinen-4-ol with antibiotic did not appear to greatly influence passage number.The susceptibility of multiply passaged S.aureus 刀 MICs,with median MICs obtained under the three conditions NCTC6571 to tetracycline reverted to 0.25 ug/ml after the organ- being either the same or differing by one dilution only on each ism was stored at-80C and then recultured.MICs for S.aureus day.The exception was ciprofloxacin with terpinen-4-ol,where ATCC 25923 passaged in 0.1%TTO did not differ by more than 1 the median MIC was 4-fold higher than that of ciprofloxacin alone dilution from that of the control.Passaging in TSB alone did not on days 3 and 5.The analysis of MICs showed significant differ- produce significant changes in MICs,as susceptibility data for the ences between the three conditions for ciprofloxacin on day 1(P< passaged and nonpassaged controls did not vary by more than 1 0.0001),for kanamycin on days1(P<0.0001),2(P<0.0001), dilution for all three strains (data not shown). and 6(P=0.0288),and for ampicillin on day 6(P=0.0383).For tea tree oil and terpinen-4-ol alone,the median MIC increased DISCUSSION 2-fold during the 6 days. There are many examples in the literature of the presence of a Lastly,using a macrodilution method,S.aureus strains did not second antimicrobial agent or nonantibiotic drug preventing or 912 aac.asm.org Antimicrobial Agents and Chemotherapytistical analysis of MICs obtained on each day under the three different conditions (antibiotic alone, with tea tree oil, or with terpinen-4-ol) demonstrated significant differences for cipro- floxacin on days 1 (P  0.0001) and 2 (P
0.02), for vancomycin on days 2 (P  0.0001), 4 (P
0.0017), and 6 (P  0.0001), and for mupirocin on day 2 (P
0.0082). For E. coli, increases in the MIC of more than two doubling dilutions occurred for all three antibiotics alone on days 2 to 3 (Table 3). Increases in median MICs from days 1 to 6 for antibiotic alone were 16-fold for ciprofloxacin and kanamycin and 8-fold for ampicillin. Similarly to S. aureus, the presence of TTO or terpinen-4-ol with antibiotic did not appear to greatly influence MICs, with median MICs obtained under the three conditions being either the same or differing by one dilution only on each day. The exception was ciprofloxacin with terpinen-4-ol, where the median MIC was 4-fold higher than that of ciprofloxacin alone on days 3 and 5. The analysis of MICs showed significant differ￾ences between the three conditions for ciprofloxacin on day 1 (P  0.0001), for kanamycin on days 1 (P  0.0001), 2 (P  0.0001), and 6 (P
0.0288), and for ampicillin on day 6 (P
0.0383). For tea tree oil and terpinen-4-ol alone, the median MIC increased 2-fold during the 6 days. Lastly, using a macrodilution method, S. aureus strains did not grow consistently in concentrations greater than 0.1% terpinen- 4-ol after 18 to 20 passages, demonstrating that resistance to terpinen-4ol could not be induced in vitro (Table 4). Similarly, S. epidermidis ATCC 12228 would not grow at concentrations above 0.2% terpinen-4-ol, and S. aureus ATCC 25923 would not grow above 0.1% tea tree oil. Serial passage with terpinen-4-ol resulted in few changes in antimicrobial susceptibility (Table 1). Changes in MICs of two or more dilutions were evident for ciprofloxacin, gentamicin, tetracycline, and benzalkonium chloride only. How￾ever, with the exception of benzalkonium chloride and S. aureus ATCC 25923, differences were not observed consistently for every passage number. The susceptibility of multiply passaged S. aureus NCTC 6571 to tetracycline reverted to 0.25 g/ml after the organ￾ism was stored at 80°C and then recultured. MICs for S. aureus ATCC 25923 passaged in 0.1% TTO did not differ by more than 1 dilution from that of the control. Passaging in TSB alone did not produce significant changes in MICs, as susceptibility data for the passaged and nonpassaged controls did not vary by more than 1 dilution for all three strains (data not shown). DISCUSSION There are many examples in the literature of the presence of a second antimicrobial agent or nonantibiotic drug preventing or TABLE 3 Summary of E. coli MICs of antibiotics (g/ml) alone, antibiotics with tea tree oil (0.062%) or terpinen-4-ol (0.031%), and tea tree oil or terpinen-4-ol without antibiotics, determined by serial subculturea Agent (no. of isolates) and parameter Treatment MIC on day: 123456 CIP (12) Median Alone 0.008 0.016 0.016 0.03 0.03 0.12 With TTO 0.008 0.016 0.03 0.06 0.06 0.12 With T4ol 0.008 0.016 0.06 0.06 0.12 0.12 GM Alone 0.010 0.025 0.034 0.070 0.070 0.140 With TTO 0.007 0.014 0.029 0.045 0.064 0.122 With T4ol 0.009 0.018 0.048 0.078 0.100 0.147 KAN (11) Median Alone 8 32 16 64 64 128 With TTO 4 16 32 32 32 64 With T4ol 4 16 32 32 32 64 GM Alone 6.7 45.3 20.2 60.4 50.8 107.6 With TTO 4.8 12.7 33.9 38.1 42.7 47.9 With T4ol 2.5 15.1 28.5 33.9 32.0 53.8 AMP (10) Median Alone 2 4 8816 16 With TTO 2 4 8816 16 With T4ol 2 4 8816 32 GM Alone 2.1 4.9 8.0 10.6 11.3 16.0 With TTO 2.1 4.6 8.6 9.8 12.1 12.1 With T4ol 2.1 4.9 11.3 11.3 17.1 24.3 Tea tree oil (18) Median Alone 0.5 11111 GM Alone 0.65 0.73 0.96 1.00 1.12 0.96 Terpinen-4-ol (18) Median Alone 0.12 0.25 0.25 0.25 0.25 0.25 GM Alone 0.13 0.17 0.22 0.29 0.25 0.26 a GM, geometric means. Boldface type indicates that the MIC is more than double the baseline (day 1) value. Single underlining indicates that values differed significantly from antibiotic alone on that day. Double underlining indicates significant differences between tea tree oil and terpinen-4ol treatments on that day. Hammer et al. 912 aac.asm.org Antimicrobial Agents and Chemotherapy on May 12, 2015 by SHANGHAI JIAOTONG UNIVERSITY http://aac.asm.org/ Downloaded from