Introduction of biocides into clinical practice and the impact on antibiotic-resistant bacteria
1. Summary, 121S 2. Introduction, 121S 2.1 Historical, 121S 2.2 Bacterial susceptibility or resistance to antibiotics and biocides, 122S 3. Possible linked biocide-antibiotic resistance, 123S 4. Introduction of biocides into clinical practice and bacterial resistance, 124S 4.1 Bacterial resistance to cationic biocides, 124S 4.2 Bacterial resistance to other biocides, 126S 4.3 Biocide usage and antibiotic resistance in Gram-negative bacteria, 127S 4.4 Biocide usage and antibiotic resistance in staphylococci, 127S 4.5 Biocide usage and antibiotic resistance in mycobacteria, 128S 5. Overall comments and conclusions, 129S 6. References, 130S Biocides and other antimicrobial agents have been employed for centuries. Much later, iodine found use as a wound disinfectant, chlorine water in obstetrics, alcohol as a hand disinfectant and phenol as a wound dressing and in antiseptic surgery. In the early part of the twentieth century, other chlorine-releasing agents (CRAs), and acridine and other dyes were introduced, as were some quaternary ammonium compounds (QACs, although these were only used as biocides from the 1930s). Later still, various phenolics and alcohols, formaldehyde and hydrogen peroxide were introduced and subsequently (although some had actually been produced at an earlier date) biguanides, iodophors, bisphenols, aldehydes, diamidines, isocyanurates, isothiazolones and peracetic acid. Antibiotics were introduced clinically in the 1940s, although sulphonamides had been synthesized and used previously. After penicillin came streptomycin and other aminoglycosides-aminocyclitols, tetracyclines, chloramphenicol, macrolides, semi-synthetic beta-lactams, glycopeptides, lincosamides, 4-quinolones and diaminopyrimidines. Bacterial resistance to antibiotics is causing great concern. Mechanisms of such resistance include cell impermeability, target site mutation, drug inactivation and drug efflux. Bacterial resistance to biocides was described in the 1950s and 1960s and is also apparently increasing. Of the biocides listed above, cationic agents (QACs, chlorhexidine, diamidines, acridines) and triclosan have been implicated as possible causes for the selection and persistence of bacterial strains with low-level antibiotic resistance. It has been claimed that the chronological emergence of qacA and qacB determinants in clinical isolates of Staphylococcus aureus mirrors the introduction and usage of cationic biocides. Biocides (antiseptics, disinfectants and preservatives) and other antimicrobial agents have been used in various forms for centuries. Early empirical approaches used copper and silver vessels for storing potable water; vinegar and honey for cleansing wounds; balsams as natural preservatives in aiding mummification; and drying, salting and spices for preserving fish and meat. Fracastoro's concept of the `seeds of disease' as possible aetiological agents was put forward in the fifteenth century and the `animalcules' of van Leeuwenhoek described in the seventeenth. Later, a method of quantifying chemical preservation was devised by Pringle, in which solutions of various salts were compared with a standard (sea salt) in terms of their relative ability to preserve lean meat. Later still, reports were made about the use of iodine as a wound disinfectant, of chlorine water in obstetrics and of phenol (carbolic acid) as a wound dressing and in antiseptic surgery (the high toxicity of agents applied directly to the human body is to be noted), and of the claimed sporicidal activity of mercuric chloride. These and other aspects of the early historical development of these chemical agents are well reviewed by 58, 59, 61, 62) and 21). In the early part of the twentieth century, other chlorine-releasing agents (CRAs) and some quaternary ammonium compounds (QACs) were introduced (see 61, 63; 21). By 1945, the biocides in common use included phenolics, organomercurials, CRAs, iodine, alcohols, formaldehyde, hydrogen peroxide, silver compounds and dyes (acridines, triphenylmethanes). Several of these remain in use at the beginning of the twentyfirst century. Some of the biocides introduced since 1945 (122) had actually been produced at an earlier date but not tested for antimicrobial efficacy until some time had elapsed. Probably the most important agents introduced since 1945 are biguanides (chlorhexidine, alexidine and polymeric forms), amphoteric surfactants, bisphenols including triclosan, aldehydes (notably glutaraldehyde, succinaldehyde-based products and ortho-phthalaldehyde), CRAs such as isocyanurates, iodine-releasing agents (iodophors), isothiazolones and peracetic acid (Table 1). Although antibiotics are often classically considered as dating from Fleming's work in the 1920s, in fact, the curative value of moulds has been known since ancient times and work in the nineteenth century was undertaken on the antibacterial activity of Penicillium spp. by Burdon, Sanderson and especially by Joseph Lister, William Roberts and John Tyndall (129). In some instances, a mould was even used to treat human infections. Later, it was shown (see 130) that several bacterial species could produce β-lactamases with the ability to hydrolyse some, but not necessarily all, types of β-lactams. After benzylpenicillin came streptomycin, the tetracyclines, chloramphenicol, the newer, semisynthetic penicillins, the cephalosporins and many other antibiotics (Table 2), including antitubercular drugs. By design or otherwise, research on antibiotics and nonantibiotic antimicrobial agents has tended to proceed separately (123). Isolated reports appeared in the 1970s (e.g. 116) about the susceptibility to biocides of antibiotic-resistance bacteria and it was recognized (44) that mercury resistance is inducible and plasmid-borne and is transferable by conjugation or transduction. Further, inorganic (Hg2+) and organomercury resistance is common in clinical isolates of Staphylococcus aureus that carry penicillinase plasmids (131; 99). Plasmids in Gram-negative bacteria may also carry genes that confer resistance not only to antibiotics but also, in some cases, to cobalt (Co2+), nickel (Ni+), cadmium (Cd2+) and arsenate (AsO3–4) (132), not, of course, that these can normally be considered as `true' biocidal agents. Reduced susceptibility, or adaptation, to biocides was described by 96) and 52, 53). In the 1980s, increasing interest developed in the relative responses of Gram-positive bacteria, especially staphylococci, and Gram-negative organisms (notably Pseudomonas spp., Proteus spp., Providencia stuartii) to biocides and antibiotics from which a possible linkage was the logical outcome. Biocides to which bacterial resistance might be a problem are shown in Table 3, and general mechanisms of resistance in Table 4. There has been considerable debate as to whether biocide resistance and antibiotic resistance are in any way linked. At first sight, the two groups of antibacterial agents are quite dissimilar. Antibiotics (31) are generally considered as being selectively toxic agents suitable for administration to patients, whereas biocides have traditionally been regarded as antiseptics, disinfectants or preservatives. Additionally, antibiotics are thought of as having a specific target site within a bacterial cell and biocides as having multiple target sites (63). This latter concept has received something of a jolt with the finding (83), since amply confirmed, that enoyl reductase is a primary target site for the action of the bis-phenol (phenylether), triclosan, in Escherichia coli. It must be added that the resulting inhibition of fatty acid synthesis is unlikely to be the only effect possessed by triclosan. There are also similarities in the actions of some antibiotics and biocides. These can be envisaged as involving uptake into Gram-negative bacteria by a self-promoted entry system, inhibition of enoyl reductase in mycobacteria by isoniazid as well as triclosan and filament induction in Gram-negative cells (Table 5). General mechanisms of bacterial resistance to antibiotics and biocides are presented in Table 4. From this, it is clear that similarities and differences exist in the ways in which bacteria can overcome the actions of the two groups of antibacterial agents. Thus, for both, two major mechanisms of resistance are known, intrinsic and acquired. Impermeability, target site modification, enzymatic inactivation and the increasingly important efflux systems can, in general terms, nullify or reduce the action of antibiotics and biocides. However, specific mechanisms obviously apply to individual members of both groups. Multidrug (antibiotic) resistance is a major clinical problem (50). Several issues may be raised in consequence: (i) do antibiotic-resistant bacteria remain sensitive to biocides; (ii) are biocide-resistant bacteria also resistant to antibiotics; (iii) can biocides select for antibiotic-resistant bacteria; and (iv) can the introduction of biocides into clinical practice have an impact on antibiotic resistance? The first two questions can be answered quite simply, because it has been shown (reviewed by 119,120) that antibiotic-resistant bacteria are not generally more resistant to in-use biocide concentrations than the corresponding sensitive bacteria. Bacteria showing reduced susceptibility to biocides may or may not be more resistant to antibiotics (143, 144, 145; 141) and any increased drug resistance might be associated with a nonspecific increase in outer membrane permeability. As to the third question, biocides such as pine oil disinfectant (88) and triclosan (84) can select for low-level resistance to antibiotics in E. coli by the action of a multidrug efflux pump and exposure to triclosan of triclosan-sensitive mutants of Ps. aeruginosa produces a concomitant huge increase in resistance to ciprofloxacin (33). The mutants overexpressed a multidrug efflux pump, MexCD-OprJ. The clinical significance of this laboratory finding is unclear. It does, however, raise concerns about the fourth issue raised above, i.e. whether the introduction into clinical practice of particular biocides in the widest sense (the chronological date in which they were first used: Section 2) or the narrower sense (first use in a particular hospital) has any role to play in the selection and development of antibiotic-resistant bacteria. This issue is explored more fully below (Section 4). Although bacterial resistance to biocides is considered to be a recent problem, in actual fact reports appeared several years ago in which laboratory and environmental resistances were described. 27,28), for example, trained Ps. aeruginosa to grow in high concentrations of a QAC, although in retrospect it seems rather odd that high concentrations of QAC in excess of 1000 p.p.m. (1000 μg ml–1) could be attained in a nutrient liquid medium. At about the same time, 78) showed that Ps. pyocyanea (aeruginosa) could contaminate cetrimide and other fluids. This finding has since been amply confirmed by other authors (71; 5; 6, 7 ; 109; 80; 74; 86; 26; 1; 19; 49; 125; 18; 25; 98; 107; 135; 57; 41; 45; 70; 89; 42; 127; 66; 69). In most of these publications, Ps. aeruginosa and Ps. multivorans (later Ps. cepacia, now Burkholderia cepacia) were implicated as contaminants of benzalkonium chloride, other QACs or chlorhexidine. However, it would be incorrect to conclude from such studies that true bacterial insusceptibility was in many instances, inactivation of a QAC disinfectant had from the of was employed at a of in μg ml–1) some of the undertaken to be of water as the use of for Several authors that the introduction of more the problem of disinfectant Further, such in more of the and of disinfectant In it is of that some authors the antibiotic of organisms that had been from disinfectant solutions and that had of or resistance could be in fact, made the that might have been from the in their disinfectant in by the antibiotics that were at the resistance in that clinical exposure to the was by found that whereas of standard strains of were μg clinical isolates of this from to μg These resistant strains not, however, resistance to or and only reduced susceptibility to two antibiotic-resistant has been for a aspects of resistance are described by and In (Table resistance to cationic biocides, such as chlorhexidine, and has been resistance is by several multidrug resistance determinants (Table of which the is the most These determinants to other found in bacteria The is by the of the These multidrug efflux can efflux many and biocides as well as dyes such as by a human or is the efflux to many is by is a of the of of the multidrug resistance have been found in various other types of including some bacteria such as E. coli However, many multidrug efflux systems are by the rather than by as is by the to efflux a of compounds as well as and antibacterial agents. The other multidrug efflux systems are Gram-positive and Gram-negative bacteria and to of the multidrug resistance the major and the In (Table the genes qacB and known as and confer resistance to cationic biocides and to dyes such as The qacA and qacB genes part of the are found on whereas the may be on both and plasmids in aureus and The to the that also the and the The was on a and is a of and toxic has also been efflux are found in the or groups The are to Gram-negative bacteria and work in with a membrane and an outer membrane efflux systems associated with Ps. aeruginosa are presented in Table also and As in Section several biocides were in common use in the early years of the century. In of their a of many it would be that bacteria resistant to their action would have in the and have shown that strains to known preservatives have The to which this is a problem is quite of the of to in in concentrations although might not be actual in-use concentrations are are not suitable for antiseptic and generally as only a in biocidal biocidal causing some is triclosan resistance has been found in E. coli and aureus strains 4.3 and strains with low-level resistance μg ml–1) to triclosan from with and triclosan these strains were also μg resistance was transferable but included However, 141) not in triclosan associated with the of a resistance. about triclosan resistance have been also of resistance to other biocides has been to isothiazolones in and to in the clinical may play a major role isolates of Gram-negative bacteria to be to a of biocidal agents than do standard for this is the by use of particular biocides. to be considered is the within a at which isolates are Thus, isolates from tended to be Gram-negative bacteria that were to inactivation by biocides than from a The as to whether such isolates reduced susceptibility to antibiotics associated with this, whether antibiotic resistance has from the introduction of biocides into and found that the use of in the of in by in the of strains of Ps. and with increased resistance to and also resistance to to It was that the was as a of to a of antibiotic and resistance were studies with strains of Ps. that exposure to the in susceptibility with increased nonspecific resistance to some antibiotics also being found However, in studies with other Gram-negative bacteria have often to any or QAC insusceptibility and antibiotic resistance. Of possible significance are the of bacteria from especially in of the that and of water may the of antibiotic-resistant bacteria. was as being for or such of or in laboratory or clinical strains of E. coli their susceptibility to triclosan increasing about to to antibiotics was also However, these do not necessarily that the use of triclosan in clinical practice or in the is for for antibiotic resistance. the finding that exposure to triclosan of a triclosan-sensitive Ps. aeruginosa on an efflux pump that the cells resistant to ciprofloxacin has not as been to the clinical can the introduction of triclosan into clinical practice in the 1970s as a be for resistance to in the Bacterial resistance to the is to (although the of is reduced to the in outer and efflux. The on the of entry of these outer membrane and the of an efflux used disinfectant is pine found that E. coli mutants for resistance to pine oil the and showed low-level resistance to and acid. mutants which also overexpressed for resistance to pine and of resistance to and the other antibiotics than the The is by and and produced a increase in susceptibility of E. coli to pine oil and also to a QAC and aureus is a major of in in the and although not strains increased Staphylococcus aureus in the for with some strains being in their and of high strains are that in the being and It is that strains have by the same mechanisms of and that in other The emergence of resistance plasmids the of This is also considered to be for the of resistance plasmids There are strains of aureus that the use of the but the of the has from selection by the use of the It is also that the emergence of biocide-resistant strains has in a The of aureus strains resistant to more than antimicrobial agents has been and plasmids cadmium and biocide resistance described It is to and to the introduction of and disinfectants into clinical practice has been for the of antibiotic-resistant strains of The concept of efflux systems as being general mechanisms for has been studies with the In that antiseptic efflux not However, the qacA is not found in strains In that resistance in various was associated with other resistance to a of from an and of the of resistant strains in was by the use of antibiotics or It was that both types of agents could select strains that were resistant to or both antibacterial groups. also, have that cationic biocides such as QACs as a such that the of multidrug efflux from the of genes these The qacA and genes might have from genes for the and systems a common and ancient that the introduction into clinical practice of cationic biocides it is that the introduction of cationic biocides could have for strains such This a of aureus a multidrug resistance the qacB The emergence of the qacA in aureus isolates the that qacA had from and that the use of and was It is rather to the latter to in this because it is not used in and not as a biocidal on to that a QAC, benzalkonium chloride, the of qacA and qacB and that their chronological emergence in clinical isolates of aureus the introduction and usage of cationic biocides in the diamidines, QACs chloride, and chlorhexidine. Of the have not been used for many years in the at the and QACs are not used to any great although QACs may part of some biocidal It be possible to in the research of aureus isolates or genes to high or concentrations of or a QAC other cationic could be undertaken to whether increased resistance would to the and to other biocides with a concomitant in antibiotic resistance. The (phenylether), triclosan, has been used in products for years including and body as well as use has to concerns being that triclosan could a for antibiotic-resistant strains of other bacteria, 4.3 and and mutants of aureus have been from within inhibition increased resistance was to a of antibiotics in this and in this and in the studies of and have of isolates some for their or resistance to triclosan and to cationic biocides. The ml–1) for isolates were and the being and μg Some of the strains had triclosan of μg and had of μg These are to presented some years earlier by and that have in the of strains to triclosan in the have been known to be to biocides than other bacteria The most important for this is the of the cell There is that biocide efflux is or that resistance can be of to streptomycin was first found many years ago and resistance to other antitubercular has also been described problem of recent times has been the increasing of a that is as resistance to isoniazid and with or resistance to other drugs. to or streptomycin is of clinical is that biocides such as QACs or are for for strains and it is unlikely that such an would the possible triclosan and isoniazid to be explored acid was introduced in for the of action is that of a by a of the into an of to the whereas of the from clinical isolates of produced resistance. by the is in acid As have that triclosan an enoyl reductase in E. coli. It has also been shown that in the of in resistance to both triclosan and The of the in which mutants by triclosan showed resistance to raised the that triclosan could for the emergence of It is to that is and that low-level resistance in is associated with or within the whereas resistance is linked to major in the with the of activity in the of confer a of resistance that is not clinically of by and has been described the enoyl reductase from E. coli The enoyl from is to and is to the triclosan and in and it is that triclosan in a to and however, two and found in clinical isolates of sensitive to triclosan (Table from which it was that at the enoyl site could be strains of this From the clinical it as to whether the introduction of triclosan as an antimicrobial some years ago has a role in the development of There is that isolates to be to biocides than for this is the usage of biocides, which as a selection in the isolates from of a in and also in to biocides. this were the only it would be that bacterial strains from sites in which biocides are actually would also a high resistance to such agents but this is not necessarily example, in a triclosan Ps. aeruginosa strains as and some strains of aureus concentrations of triclosan for inhibition However, from of and a both of which were other strains were found and it was not possible to triclosan resistance with antibiotic resistance. From the is some that the introduction of biocides into clinical practice might have an impact on antibiotic resistance. This into in to the by antibiotics in the of human and and as a of their into of the of biocides and antibiotics of isolates from or more years are but only to a can about in resistance but do not necessarily any such biocide introduction or is is a of the of isolates the 1950s to the time linked to a of the introduction of specific biocides at known times this of the strains and of the in resistance to biocides, antibiotics and to both groups would an important part of such a that to be is the possible role that biocide may have on the development of biocide resistance. studies to date involving and exposure of Ps. aeruginosa to cationic biocides have not any that this is a problem but is have that Ps. aeruginosa is of resistance rather than isothiazolones as a of their environmental but not whether in antibiotic susceptibility has to be is such as which might to or triclosan are than corresponding strains with Biocides have a of usage they have a role to play in in the and especially in the also have important in both the disinfectant and in the and other types of biocides and antibiotics were presented in and with about their introduction into Of the biocides in particular have been as being associated with antibiotic resistance, chlorhexidine, QACs and triclosan and and In bacteria may resistance to biocides by outer membrane that may increase insusceptibility to Biocide may but significance in to in-use concentrations is more is that found from the of multidrug efflux that may be for antibiotic resistance and low-level biocide In efflux of has been described but is that this is linked to biocide resistance. However, in E. coli of multidrug efflux pump multidrug resistance and increased susceptibility to several biocides and antibiotics In laboratory studies have shown some of biocide and antibiotic There have been that the introduction of biocides into clinical practice has been for the selection of antibiotic-resistant bacteria this is from being the possible linkage bacteria and antibacterial resistance to be explored