Antiseptics: New Problems and a Few New Solutions
Abstracts & Commentary
Synopsis: A single integration event is sufficient for the introduction of a gene associated with reduced susceptibility of K pneumoniae to chlorhexidine. Increasing bacterial resistance to antiseptic agents demonstrates the need for novel agents, such as the essential oil of the tea tree.
Sources: Fang CT, et al. Cloning of a cation efflux pump gene associated with chlorhexidine resistance in Klebsiella pneumoniae. Antimicrob Agents Chemother. 2002;46:2024-2028; Carson CF, et al. Mechanism of action of Melaleuca alternifolia (tee tree) oil on Staphylococcus aureus determined by time-kill, lysis, leakage, and salt tolerance assays and electron microscopy. Antimicrob Agents Chemother. 2002;46:1914-1920.
Fang and colleagues at the National Taiwan University Hospital examined 50 randomly collected clinical isolates of Klebsiella pneumoniae in an attempt to isolate the gene or genes responsible for chlorhexidine resistance in this organism. The chlorhexidine MICs of these isolates ranged from 4 mg/mL to greater than 32 mg/mL; the MIC of 30 (60%) of the isolates was > 32 mg/mL. A phagemid (see Table for definition) expression library containing genomic DNA from a chlorhexidine-resistant strain was created in a standard E coli strain (XLORL). The library yielded 20 clones that grew on agar plates containing chlorhexidine in a concentration of 16 mg/mL.
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DNA sequencing of these clones revealed that each contained an identical 903-nucleotide locus with a 90% similarity to yiip, a gene coding for a putative transporter in E coli K12. Because the deduced amino acid sequence suggested that the gene product probably functions as a cationic efflux pump, the gene, which was demonstrated to be present on the chromosome, was designated cepA. When inserted into E coli, cepA encoded a 33-kDa protein that was associated with a 2-4-fold increase in chlorhexidine MIC.
Thus, a single integration event is sufficient for insertion of cepA into the genome of Gram-negative bacteria and the expression of cepA is associated with a doubling or quadrupling the chlorhexidine MIC.
Despite a concern about reduced efficacy of topical antimicrobial agents, few truly new products have been introduced in recent decades—as in the world of oncology, microbiologists are evermore turning to the natural world to find new products. Malaleuca alternifolia (tee tree) oil is a good example. The oil of the tea tree is a complex blend of alcohols, many of which have already been shown to have good antibacterial activity in a series of studies by Australian microbiologists. In their current work, Carson and associates studied the antistaphylococcal activity of 3 components of tea tree oil: 1,8-cineole, terpinen-4-ol, and alpha-terpineol.
The MICs of these compounds are good, expressed as percentages, around 1% or less. Killing of S aureus was studied using one half the MIC, the MIC, and 2 times the MIC. Each of these concentrations were active and the most bactericidal was the compounds terpinen-4-ol and alpha-terpineol. Indeed, they rapidly reduced a 1010 inoculum to 102 within 30 minutes or faster. Bacteriolysis was one mechanism as demonstrated by the loss of absorbing material at 620 nm, an effect that lasted as long as 23 hours.
Tea tree oils also caused loss of salt tolerance, a central property of S aureus. This loss of salt tolerance was seen with as little as 0.25 the MIC and was profound at 2´ the MIC, particularly with 1,8-cineole.
EM visualization showed membrane damage with the presence of mesosomes and loss of cytoplasmic material, effects produced also by vancomycin, betanes, defensins, and some other alcohols.
Comment by Joseph F. John, Jr., MD
What we are not told by Fang et al is how much of a clinical problem chlorhexidine-resistant K pneumoniae isolates have become in Taiwan. We are also not told about the presence of cepA in other Gram-negative bacteria in Taiwan and environs. What we do know from the study is that an acquired chromosomal chlorhexidine resistant gene in a common Gram-negative pathogen like K pneumoniae has become stable in many isolates in Taiwan. CepA is likely functioning as an efflux pump similar to another acquired pump in S aureus known as qac varieties qacA and qacB. Indeed, gene mining techniques showed that cepA is similar to a transmembrane efflux protein found in S enterica, so this type of gene may be even more broadly distributed in nosocomial, as well as community pathogens, a scary thought for sure.
The prospect of cepA spread is scary since we depend on the ability of the residual concentration of antiseptics like chlorhexidine to kill bacteria on the skin. In fact, 99.9% of killing will occur with only 10-50 times the MIC so that the mere increase of the MIC from 2 µg/mL to 16 µg/mL could lead to persistence of organisms like K pneumoniae on the hands.
What are the prospects for new antiseptics? For years, natural agents like tea trea oil have been touted for their contact antibacterial properties. Australian scientists and physicians have been the most enthusiastic about tea tree oil as a topical antimicrobial. Carson et al at the University of Western Australia have already written several provocative papers including one describing the use of tee tree oil as an alternative to mucosal decolonization of methicillin-resistant S aureus.1
Their most recent contribution involves more proof that tea tree oil has excellent antistaphylococcal activity with certain components, particularly alpha-terpineol or terpinen-4-ol, acting within 30 minutes. Besides their lytic potential, tea tree oils also reduce salt tolerance in S aureus, suggesting that osmoregulation may be hampered. There may be additional mechanisms of action of these lipophilic biocides. Tea tree oil may exhibit such potent antibacterial activity due to multiple mechanisms resulting from its complex makeup of at least 100 terpenes and other related alcohols.
So, these 2 articles suggest that there is a potential problem with currently used antiseptics, like chlorhexidine, namely resistance. The epidemiology of chlorhexidine resistance, particularly that spawned by the efflux pump, cepA, remains to be defined; but using cepA as a DNA probe, that data should be rapidly forthcoming. For those hospitals facing what they feel are real problems of chlorhexidine resistance, they have few alternatives. We were faced with such an apparent problem back in the 1980s in a neonatal ICU and resorted to the use of hexachlorophene to quell the outbreak,2 but such a maneuver can raise other problems. More recently, mupirocin has become a staple for nasal decolonization, hampered already by widespread resistance in some parts of the world.
Clearly, new and improved antiseptics are needed to fight the expanding problem of resistance to topical agents. The hysteria created by the media that we are drowning in a sea of pathogens has resulted in widespread use of antibacterial soaps for residential use. Such widespread use can only complicate the problem of resistance in organisms that routinely live comfortably on our skin and in mucosal membranes.
Also of concern is the potential for induction of cross-resistance to clinically useful antibacterial agents by the widespread use of topical antiseptics. Chlorhexidine resistance in S aureus has been reported to be related to selection of staphylococci containing qacA genes, which encode an efflux system, in multiresistance plasmids.3
In addition, triclosan resistance in P aeruginosia has been reported to be associated with a multidrug-resistant phenotype and with a 94-fold increase in ciprofloxacin MIC.4,5
Dr. John, Chief, Medical Subspecialty Services, Ralph H. Johnson Veterans Administration Medical Center; Professor of Medicine, Medical University of South Carolina, Charleston, SC, is Co-editor of Infectious Disease Alert.
References
1. Caelli M, et al. Tea tree oil as an alternative topical decolonization agent for methicillin-resistant Staphylococcus aureus. J Hosp Infect. 2000;46:236-237.
2. Reboli AC, et al. Epidemic methicillin-gentamicin-resistant Staphylococcus aureus in a neonatal intensive care unit. Am J Dis Child. 1989;143:34-39.
3. Russell AD. J Pharm Pharmacol. 2000;52:167-173.
4. Chuanchuen R, et al. Antimicrob Agents Chemother. 2001;45:428-432.
5. Deresinski S. Antiseptics and antibiotic resistance. Infectious Disease Alert. 2001;20(10):76-77.
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