Aromatic alcohols and their effect on Gram-negative bacteria, cocci and mycobacteria

Sir, Phenethyl alcohol (2-phenylethanol; PEA, mol. wt 122.17) inhibits a range of Gram-negative bacteria, but not Pseudomonas fluorescens, at a concentration of 0.2% (2000 mg/L), whereas Gram-positive cocci, such as Staphylococcus aureus are inhibited at 0.5% w/v (5000 mg/L), with Enterococcus faecalis requiring still higher concentrations.1 PEA-containing media had earlier been suggested as a means of selecting for Gram-positive bacteria in mixed flora.2 Interestingly, Mycobacterium smegmatis and Mycobacterium phlei were also inhibited at 0.2%, suggesting that the solubility of PEA in lipids could play a major role in its selective action.1 However, Wilson et al.3 reported that PEA was only slightly less inhibitory to Staphylococcus aureus than Escherichia coli, and that the MIC for Pseudomonas aeruginosa was 0.46% (4600 mg/L). As part of comprehensive studies of the responses to biocides of mycobacteria, other Gram-positive bacteria and Gram-negative organisms, it was decided to investigate the effects of PEA and of another, significantly more hydrophobic, aromatic alcohol (5-phenyl-1-pentanol; 5-PP, mol. wt 164.25) on some strains of Mycobacterium chelonae, M. smegmatis and Burkholderia cepacia. B. cepacia K56-2 triclosan- and chlorhexidine-susceptible mutants were isolated using a Tn5-pOT182 random mutagenesis system. Strain K56-2 is a genetically amenable clone of the genome sequencing strain J2315. A single strain used in disinfectant testing [EN 1276 (1997)] of each of E. coli, P. aeruginosa and S. aureus was included for comparative purposes. M. chelonae NCTC 946 is a glutaraldehyde-susceptible control, whereas M. chelonae strains Epping and Harefield are glutaraldehyde resistant but ortho-phthalaldehyde susceptible.4M. smegmatis LIMP7 is a derived mutant of M. smegmatis mc2155 with an altered cell envelope permeability. This mutant has a defective impA1 (inositol mono-phosphate phosphatase) gene, leading to a dramatic decrease in lipoarabinomannan synthesis. MIC determinations were undertaken in triplicate by means of the Denley multipoint inoculator with an inoculum size of ∼105 cfu in 1 µL delivered. Incubation temperature was at 30°C (M. chelonae strains) for 3–4 days, 37°C (M. smegmatis mc2155 for 3–4 days, S. aureus and Gram-negative bacteria for 2 days) and 42°C (LIMP7 for 3–4 days). At 37°C, the LIMP7 mutant reverts to the parental strain due to the genomic insertion of a temperature-sensitive transposon. MICs of PEA and 5-PP for Gram-positive and -negative bacteria are presented in Tables 1 and 2, respectively. As noted previously, PEA was less active against S. aureus than against E. coli or mycobacteria, and its MIC for P. aeruginosa was also high. Glutaraldehyde-resistant mycobacteria (strains Epping and Harefield) had the same response to both PEA and 5-PP as the susceptible strain (NCTC 946). This is an interesting observation, because some M. chelonae strains trained in the laboratory to high glutaraldehyde resistance are believed to show alterations in the mycoylarabinogalactan complex of the cell wall.5 All of the B. cepacia strains showed the same order of response to PEA, whereas two strains (ER10-11 and ER17-15, especially the former) were more susceptible than the other strains to 5-PP. The LIMP7 mutant strain was more susceptible than the wild-type to PEA, but this could be due, at least partly, to the increase in incubation temperature potentiating the activity of this alcohol. 5-PP was considerably more effective than PEA against any strain, although with P. aeruginosa no inhibition was achieved at its maximum solubility. The log (p) [n-octanol/water] value (Crippen’s fragmentation) of PEA is +1.74 and of 5-PP is +2.99. conclusion: (i) apart from P. aeruginosa, PEA shows greater activity against Gram-negative bacteria and mycobacteria than against the S. aureus strain; (ii) at its maximum solubility, 5-PP does not inhibit the growth of P. aeruginosa; (iii) inhibitory concentrations of PEA and 5-PP are ∼1.5–2.6 and 1.4–1.75 times higher, respectively, against S. aureus than against mycobacteria (Table 1); (iv) on a molar basis, inhibitory concentrations of PEA (Table 2) are ∼7.2 times higher than 5-PP against E. coli and ∼3.7–4.2 times greater against B. cepacia strains, except for ER10-11 (11.8 times) and ER17-15 (5.6 times); and (v) 5-PP appears to be taken up to a greater extent than PEA against all bacteria (except P. aeruginosa, for which no comparison can be made), probably because of its greater lipophilicity and damage to the cytoplasmic membrane. We wish to thank ASP Biocides, Irvine, CA, USA and the Welsh School of Pharmacy, Cardiff, UK for research studentships (to S.F. and E.L.R., respectively). Corresponding author. Tel: +44-2920-875812; Fax: +44-2920-874149; E-mail: russellD2@cardiff.ac.uk Present address. School of Pharmacy and Biomolecular Sciences, University of Brighton, Brighton BN2 4GJ, UK MIC ranges of PEA and 5-PP for Gram-positive bacteria aEN 1276 (1997) test strain. MIC ranges of PEA and 5-PP for Gram-positive bacteria aEN 1276 (1997) test strain. MIC ranges of PEA and 5-PP for Gram-negative bacteria CHX, chlorhexidine. aEN 1276 (1997) test strain. bNo activity at maximum solubility. cThis strain has been the subject of a genome sequence initiative (http://www.sanger.ac.uk/Projects/B_cepacia/) and will become the Type strain for the new species Burkholderia cenocepacia (E. Mahenthiralingam, unpublished data). MIC ranges of PEA and 5-PP for Gram-negative bacteria CHX, chlorhexidine. aEN 1276 (1997) test strain. bNo activity at maximum solubility. cThis strain has been the subject of a genome sequence initiative (http://www.sanger.ac.uk/Projects/B_cepacia/) and will become the Type strain for the new species Burkholderia cenocepacia (E. Mahenthiralingam, unpublished data). 1Welsh School of Pharmacy and 2School of Molecular and Medical Biosciences, Cardiff University, Cardiff CF10 3XF, UK