5. Does biocide use contribute to the development of antibiotic resistant bacteria?
The SCENIHR opinion states:
3.6. Linkage between biocides usage and antibiotic resistance
3.6.1. Laboratory/in vitro
There have been a number of laboratory-based investigations describing a possible linkage between biocide use and antibiotic resistance (Akimitsu et al. 1999, Braoudaki and Hilton 2004a, Braoudaki and Hilton 2004b, Chuanchuen et al. 2001, Russell et al. 1998, Tattawasart et al. 1999, Walsh et al. 2003). This concept is not novel and a number of studies indicate the possibility for such linkage following exposure to various biocides such as the bisphenol triclosan (Braoudaki and Hilton 2004a, Braoudaki and Hilton 2004b, Chuanchuen et al. 2001, McMurry et al. 1998a, Moken et al. 1997, Sánchez et al. 2005), the biguanide chlorhexidine (Kõljalg et al. 2002, Russell et al. 1998, Tattawasart et al. 1999), and quaternary ammonium compounds (Akimitsu et al. 1999, Walsh et al. 2003). In many laboratory-based studies, similar mechanisms have been implicated in resistance linkage such as impermeability (Tattawasart et al. 1999a), multi-drug efflux pumps (Levy 1992, Moken et al. 1997, Noguchi et al. 2002, Randall et al. 2007, Schweizer 1998, Zgurskaya and Nikaido 2000), over expression of multigene components or operons (Levy 1992) such as mar (McMurry et al. 1998b, Moken et al. 1997), soxRS and oxyR (Dukan and Touati 1996, McMurry et al. 1998a, Wang et al. 2001), and the alteration of a target site (McMurry et al. 1999).
The selective pressure exerted by exposure to biocides has been associated with the increasing incidence of resistance to antibiotics. For example, the use of cationic biocides has been blamed for the spread of the qac genes and thus for the widespread occurrence of multi-drug efflux pumps (Heir et al. 1998, Heir et al. 1999, Mitchell et al. 1998; Paulsen et al. 1996a, Paulsen et al. 1996b, Sundheim et al. 1998). Chlorination has been associated with a higher incidence of antibiotic resistance (Murray et al. 1984) and a number of studies have claimed a direct link between biocide exposure and antibiotic resistance (Aiello and Larson 2003, Akimitsu et al. 1999, Kunonga et al. 2000, Levy 2000, Moken et al. 1997). Another study showed that a single exposure to the preservatives sodium nitrite, sodium benzoate or acetic acid induced bacterial resistance to multiple antibiotics (tetracycline, chloramphenicol, nalidixic acid and ciprofloxacin), although clinical levels of resistance were not reached. The cross-resistance was linked to mar mutations (Potenski et al. 2003). More recently Randall et al. (2007) isolated a mutant of S. enterica showing antibiotic resistance following treatment with a low concentration of an aldehyde, oxidising, QAC or phenolic-based disinfectant. The change in the observed antibiotic susceptibility profile depended upon the disinfectant tested and the mutants isolated. Following exposure to an aldehyde-based disinfectant, isolated mutants that were resistant to ciprofloxacin exhibited either some type of efflux mechanism or a mutation in GyrA (Randall et al. 2007). The effect of biocides on the bacterial cell is complex and the emergence of bacterial cross-resistance following exposure to biocides might be strain specific rather than species or genus specific (Braoudaki and Hilton 2004b).
Other investigations have however failed to make a direct link between biocide exposure and antibiotic resistance, although the antibiotic susceptibility of the bacterial strain was altered (Lear et al. 2000, Lear et al. 2002, Nomura et al. 2004, Thomas et al. 2000, Thomas et al. 2005, Walsh et al. 2003, Winder et al. 2000). A decrease in E. coli susceptibility to triclosan following repeated exposure, but not necessarily to other Gram-negative bacteria has been reported (Ledder et al. 2006, McBain et al. 2004b). More importantly, when the decrease in susceptibility to triclosan was observed, it was not linked to a decrease in susceptibility to unrelated biocides and antibiotics.
The presence of conjugative plasmids has been associated with co-resistance between a number of biocides such as cationic compounds (Beveridge et al. 1997, Langsrud et al. 2003, Paulsen et al. 1996a) and metallic salts (e.g. organomercurials) (Misra 1992) and antibiotics.
3.6.2. Consumer products
The same or similar chemicals are sometimes used as preservatives in several household and personal hygiene products. Using the same antimicrobial agents (or similar molecules with respect to mechanism of action) in household products and personal hygiene products leads to exposing the bacterial flora on human skin and in the home environment repeatedly to certain biocides. This cumulative exposure may lead to reduced susceptibility of certain microbes to specific biocides (selected bacterial strains or acquired resistance under this selective pressure). However, currently available studies are inconclusive as to whether this type of bacterial exposure to biocides will lead to antibiotic resistance.
Biocides tend to act concurrently on multiple sites within the microorganism, and thus, resistance is often mediated by non-specific mechanisms. Efflux pumps have been shown to act on a range of chemically dissimilar compounds and have been implicated in both biocide- and antibiotic resistant bacteria. Cell wall changes by reducing permeability may also play a role in the observed resistance to biocides. The possibility of genetic linkage between genes for biocide resistance and those for antibiotic resistance has also been described (Fraise 2002). Although, the studies on antibiotic resistance to biocides used in the consumer products have focussed on some specific molecules (for example, triclosan, chlorohexidine, glutaraldehyde, p-chloro-m-xylenol, quartery ammonium compounds/benzalkonium chloride, pine oil and chlorine releasing compounds), the mechanism of actions of these molecules may also be applicable to the long list of biocides used in the consumer products.
Considerable controversy surrounds the use of biocides in an ever increasing range of consumer products and the possibility that their indiscriminate use might reduce biocide effectiveness and alter susceptibilities towards antibiotics (Aiello et al. 2005, Aiello et al. 2007, Braoudaki and Hilton 2004a and b, Gilbert and McBain 2003, McBain et al. 2003a, Pumbwe et al. 2007, Russell 2004a, Weber and Rutala 2006).These concerns have been based largely on the isolation of resistant mutants from in vitro monoculture experiments. Some of the evidence suggests that exposure to biocides may be leading to increased antibiotic resistance, but this has not yet been proven in a clinical setting (IFH 2003). Further research is needed to establish a correlation between biocide exposure(s) and development of antibiotic resistance.
3.6.3. Veterinary products
In the veterinary field, data relating to the occurence of bacterial resistance following exposure to biocides are limited. The sensitivity of 700 Gram-negative bacterial strains was tested towards four antiseptics (cetrimide, chlorhexidine, hexachlorophene, mercuric chloride) and six antibiotics (ampicillin, streptomycin, erythromycin, chloramphenicol, kanamycin and tetracycline) by Maris (1991). The statistical analysis of correlation showed high positive resistance links between antiseptics and between antiseptics and antibiotics, especially for Serratia marcescens and Alcaligenes. Likewise, the investigation of 310 Gram-positive strains isolated from milking cow udders revealed positive links between chlorhexidine usage and resistance to the five tested antibiotics (ampicillin, kanamycin, streptomycin, tetracycline, gentamycine) in Streptococcus, and between hexachlorophene and oxacillin in Bacillus (Martin and Maris 1995). These studies emphasize the need to develop research and surveillance programmes in the area of animal husbandry.
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Section 3.6. Linkage between biocides usage and antibiotic resistance, p. 44 -46
3.7. Relationship between biocide availability to bacteria and resistance selection
3.7.1. Measurement of the effects of biocides on the susceptibility to antibiotics
The effect of biocides on antibiotic susceptibility in bacteria has been measured indirectly, whereby a bacterial population is treated first with a biocide and the surviving bacteria then investigated for their susceptibility to antibiotics. To our knowledge, there has been no investigation reporting the effect on bacteria of a combined treatment with biocide and antibiotic.
A number of protocols have been used to measure antibiotic susceptibility in bacterial isolates showing resistance, tolerance or increased insusceptibility to biocides. However, the large variation in the experimental parameters used generates a question about the validity of the selected protocols. Some studies based a change in antibiotic susceptibility profile on measurement of zone of inhibition (Tattawasart et al. 1999, Thomas et al. 2005). More meaningfully, other studies used standardised antibiotic susceptibility methodologies such as those given by the British Society for Antimicrobial Chemotherapy (BSAC) or Clinical and Laboratory Standards Institute (CLSI). However a limited number of studies have looked at an increase in antibiotic insusceptibility that would be associated with treatment failure (Lear et al. 2006).
This is a complex task as there are many possible interferences/biases due to the multiplicity of proposed protocols, the failure of clear comparative methodology and criteria (reference strain, reference molecule, reference experimental assay etc.), which generate a profusion of non-comparative and exploitable results (see section 3.12).
3.7.2. Possible confounding factors in dose-effect relationships
Bacteria that are resistant to inactivation by chemical disinfectants are commonly encountered in a diverse set of aquatic environments, but this apparent resistance has most often been attributed to protection by physical means, e.g. association with particulate matter or occlusion within a biofilm. Equally important are the genotypic provision of a protective capsule or spore, as well as external abiotic factors such as chemical reaction of the disinfectant with other molecules present in the aqueous environment (Berg et al. 1982).
Thus, when studying dose-effect relationships, it is of major importance to take into account antecedent growth conditions and external factors which may dramatically influence the results. The results of the experiments performed with E. coli as a model illustrated the influence of the qualitative nature of the growth environment, the degree of nutrient limitation, the temperature and the density of the microorganism on the resistance to disinfectants (Berg et al. 1982).
The population growing more rapidly could be hypothesized as more sensitive. The temperature has a relationship with lipid fluidity in the membrane (Nikaido 2003): a less permeable membrane could retard the leakage of other small constituants (like K+) critical for viability.
3.7.3. Changes in microbiota following exposure to biocides
Microcosms have been used to reproduce complex biofilm systems found in the environment, and to investigate changes in microbial population and susceptibility following exposure to biocides (McBain et al. 2004a, Moore et al. 2008). Using a drain microcosm, it was found that the use or repeated exposure to a QAC produces little changes to the population dynamic and does not alter the susceptibility profile of the microcosm (McBain et al. 2004a). However, a more recent study highlighted a clonal expansion of Pseudomonads to the detriment of Gram-positive species following QAC exposure and a decrease in biocide susceptibility for a proportion but not all test bacteria (Moore et al. 2008). Another study investigating the change in bacterial population in activated sludge following exposure to benzalkonium chloride (a QAC) showed a population shift and a selection of Pseudomonas spp following treatment (Kümmerer et al. 2002). A more recent study investigating the effect of triclosan in the development of bacterial biofilm on urinary catheter highlighted the selectivity of the bisphenol. While triclosan inhibited Proteus mirabilis, it had little effect on other common bacterial pathogens (Jones et al. 2006)
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Section 3.7,Relationship between biocide bioavailability to bacteria and resistance selection, p. 46-47