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Source document:
SCENIHR (2009)

Summary & Details:
GreenFacts (2009)

Effects of Biocides on antibiotic resistance

4. How can bacteria become resistant to biocides or antibiotics?

4.1 How can bacteria become resistant to biocides?

The SCENIHR opinion states:

3.4.3. Mechanisms of resistance to biocides Principles

Biocides have multiple target sites against microbial cells. Thus, the emergence of general bacterial resistance is unlikely to be caused either (i) by a specific modification of a target site or (ii) by a by-pass of a metabolic process. It emerges from a mechanism/process causing the decrease of the intracellular concentration of biocide under the threshold that is harmful to the bacterium. Several mechanisms based on this principle (mode of action) have been well-described including change in cell envelope, change in permeability, efflux and degradation. It is likely that these mechanisms operate synergistically although very few studies investigating multiple bacterial mechanisms of resistance following exposure to a biocide have been performed.

The efficacy of biocides depends on a range of intrinsic and extrinsic factors, (EFSA, 2008a, Reuter 1984, Reuter 1989, Reuter 1994).

Intrinsic factors are characteristics of the biocidal agent and its application. Concentration and contact time are crucial. Furthermore, the combination of contact time and concentration determines the result in term of microbial reduction. This is called the CT concept, and within certain limits of time and concentration, there is a relationship with a defined constant characterising efficacy. Thus the same result could be obtained with a high concentration of disinfectant during a short contact time, or a lower concentration during a longer contact time. The stability of the active compounds of the biocide in the environment also influences the efficacy.

Extrinsic factors derive from the environment during application. The temperature of the environment is important, as most substances have a lower efficacy at low temperatures. The presence of proteins reduces efficacy as they interact with the substance. The mode of contact also influences the efficacy, as does the contact time (mechanical effects). The pH is another important factor. The concentration of the microorganisms, the age of the bacterial community and protection by attachment on particulate matter, and the presence of biofilms (see section play an increasingly important role. Mechanisms of intrinsic bacterial resistance to biocides

Several mechanisms conferring bacterial resistance to biocides have been described; some are inherent to the bacterium, other to the bacterial population. In addition, some of the resistance mechanisms are intrinsic (or innate) to the micro-organism while others have been acquired through forced mutations or through the acquisition of mobile genetic elements (Poole 2002a). Innate mechanisms can confer high-level bacterial resistance to biocides. In this case, the term unsusceptibility is used (see definition; section

The most described intrinsic resistance mechanism is changes in the permeability of the cell envelope, also referred to as "permeability barrier". This is not only found in spores (Cloete, 2003, Russell 1990, Russell et al. 1997), but also in vegetative bacteria such as mycobacteria and to some extent in Gram-negative bacteria. The permeability barrier limits the amount of a biocide that enters the cell, thus decreasing the effective biocide concentration (Champlin et al. 2005, Denyer and Maillard 2002, Lambert 2002). In mycobacteria the presence of a mycoylacylarabinogalactan layer accounts for the impermeability to many antimicrobials (Lambert 2002, McNeil and Brennan 1991, Russell 1996, Russell et al. 1997). In addition, the presence and composition of the arabinogalactan/arabinomannan cell wall also plays a role in reducing the effective concentration of biocide that can penetrate within mycobacteria (Broadley et al. 1995, Hawkey 2004, Manzoor et al. 1999, Walsh et al. 2001).

The role of the lipopolysaccharides (LPS) as a permeability barrier in Gram-negative bacteria has been well documented (Ayres et al. 1998, Denyer and Maillard 2002, Fraud et al. 2003, McDonnell and Russell 1999, Munton and Russell 1970, Stickler 2004). There have also been a number of reports of reduced biocide efficacy following changes in other components of the outer membrane ultrastructure (Braoudaki and Hilton 2005, Tattawasart et al. 2000a, Tattawasart et al. 2000b) including proteins (Brözel and Cloete 1994, Gandhi et al. 1993, Winder et al. 2000), fatty acid composition (Guérin-Méchin et al. 1999, Guérin-Méchin et al. 2000, Jones et al. 1989, Méchin et al. 1999) and phospholipids (Boeris et al. 2007). It must be noted that in the above mentioned examples, an exposure to biocides was followed by changes in ultrastructure related to a decrease in biocidal susceptibility, usually at a low concentration (under the MIC value).

The charge property of the cell surface also plays a role in bacterial resistance mechanisms to positively charged biocides such as QACs (Bruinsma et al. 2006). It is likely that bacterial resistance emerges from a combination of mechanisms (Braoudaki and Hilton 2005, Tattawasart et al. 2000a, Tattawasart et al. 2000b), even though single specific mechanisms are often investigated.

The presence of efflux pumps is another mechanism that has been well described in the literature. It has gained increased recognition as a resistance mechanism over the past decade. Efflux pumps decrease the intracellular concentration of toxic compounds, including biocides (Borges-Walmsley and Walmsley 2001, Brown et al. 1999, Levy 2002, McKeegan et al. 2003, Nikaido 1996, Paulsen et al. 1996a, Piddock 2006, Poole 2001, Poole 2002a, Putman et al. 2000). They are widespread among bacteria and five main classes have been identified: the small multidrug resistance (SMR) family (now part of the drug/metabolite transporter (DMT) superfamily), the major facilitator superfamily (MFS), the ATP-binding cassette (ABC) family, the resistance-nodulation-division (RND) family and the multidrug and toxic compound extrusion (MATE) family (Brown et al. 1999; Borges-Walmsley and Walmsley 2001, McKeegan et al. 2003, Piddock 2006, Poole 2001, Poole 2002b, Poole 2004).

The importance of efflux pumps in terms of bacterial resistance to biocides might be considered as modest since the increase in bacterial susceptibility to selected biocides as the results of the expression of efflux pumps is usually measured as an increase in MICs rather than as resistance to a high concentration of an active substance. Efflux pumps have been shown to reduce the efficacy of a number of biocides including QACs, phenolics parabens and intercalating agents (Davin-Regli et al. 2006, Heir et al. 1995, Heir et al. 1999, Leelaporn et al. 1994, Littlejohn et al. 1992, Lomovskaya and Lewis 1992, Randall et al. 2007, Sundheim et al. 1998, Tennent et al. 1989) notably in Staphylococcus aureus with identified pumps such as QacA-D (Littlejohn et al. 1992, Rouche et al. 1990, Wang et al. 2008), Smr (Lyon and Skurray 1987), QacG (Heir et al. 1999) and QacH (Heir et al. 1998), and in Gram-negative bacteria such as Pseudomonas aeruginosa, with MexAB-OprM, MexCD-OprJ, MexEF-OprN and MexJK (Chuanchuen et al. 2002, Morita et al. 2003, Poole 2004, Schweizer 1998) and Escherichia coli with AcrAB-TolC, AcrEF-TolC and EmrE (McMurry et al. 1998a, Moken et al. 1997, Nishino and Yamagushi 2001, Poole 2004).

The enzymatic transformation of biocides has also been described as a resistance mechanism in bacteria, notably to heavy metals (e.g. silver and copper; enzymatic reduction of the cation to the metal, Cloete 2003); parabens (Valkova et al. 2001), aldehydes (formaldehyde dehydrogenase, Kummerle et al. 1996), peroxygens (catalase, super oxide dismutase and alkyl hydroperoxidases mopping up free radicals, Demple 1996). Environmental bio-degradation of various compounds has been well-described notably among Pseudomonads and complex microbial communities. However, the importance of degradation as a bacterial resistance mechanism to "in use" concentrations (high concentrations) of biocides remains unclear. As for efflux, increased resistance following degradation of biocides has been measured as an increase in MICs but not necessarily as a decreased in lethal activity.

The modification of target sites has been described on rare occasions and does not seem to be widespread among bacteria, although there is a paucity of information on this subject. The bisphenol triclosan has been shown to interact specifically with an enoyl-acyl reductase carrier protein at a low concentration (Heath et al. 1999, Levy et al. 1999, Roujeinikova et al. 1999, Stewart et al. 1999). The modification of this enzyme has been associated with low-level bacterial resistance (Heath et al. 2000, McMurry et al. 1999, Parikh et al. 2000). It has been noted that at a high concentration triclosan must interact with other targets within the cell, the alteration of which justified the lethal effect of the bisphenol (Gomez Escalada et al. 2005b). Mechanisms of acquired bacterial resistance to biocides

The development of bacterial resistance through acquired mechanisms such as mutation and the acquisition of resistant determinants are of concern since a bacterium that was previously susceptible can become insusceptible to a compound or a group of compounds (Russell 2002a). The acquisition of resistant genes has been well described in the literature (Chapman 2003, Lyon and Skurray 1987, Silver et al. 1989, White and McDermott 2001) and it is particularly important to consider this as it might confer cross- or co-resistance on occasion (Bjorland et al. 2001, Chapman 2003, Kücken et al. 2000, Poole 2004).

However, there is little information on the effect of biocides on the transfer of genetic determinants. One study in particular highlighted that while some biocides at a sub-inhibitory (residual) concentration could inhibit genetic transfer, others increased genetic transfer efficiency (Pearce et al. 1999).

There have been some investigations on co-transfer of resistant markers in epidemic methicillin-resistant S. aureus following antibiotic treatment to decolonise patients (Cookson et al. 1991a). The authors reported that there was no evidence of increase in chlorhexidine MICs six years after the first isolation of the epidemic strains, although the strain carried a qac gene (Cookson 2005). However, this was not the case with triclosan, where clinical isolates of S. aureus showed high-level mupirocin resistance and low-level triclosan resistance (MIC 2-4 mg/L) (Cookson et al. 1991b). The authors described that resistance to both chemically unrelated compounds was transferred and cured together (Cookson 2005). Table 8  summarises the main bacterial mechanisms of resistance to biocides.

Table 8: Bacterial mechanisms of resistance to biocides Expression of genes conferring resistance

The induction of bacterial resistance mechanisms following exposure to a low concentration of a biocide has been reported in a number of studies. The mechanisms involved include the over-expression of efflux pumps (Gilbert et al. 2003, Maira-Litrán et al. 2000, Randall et al. 2007), the over-expression of multigene systems such as soxRS and oxyR (Dukan and Touati 1996) and the production of guanosine 5’-diphosphate 3’-diphosphate (ppGpp) (Greenway and England 1999) (see Table 9 ).

These mechanisms are parts of the stress-response systems in bacteria, for which more evidence is available in the literature. A decrease in growth rates and altered gene expression in Escherichia coli have been described (Brown and Williams 1985, Ma et al. 1994, Wright and Gilbert 1987) following stress conditions. Exposure to isothiazolones induced the reorganisation of metabolic processes in Pseudomonas aeruginosa (Abdel Malek et al. 2002). Moken et al. (1997) described the induction of the MDR phenotype and its relevance to cross-resistance between pine oil, triclosan and multiple antibiotics. More recently, Webber et al. (2008) showed that triclosan resistance in Salmonella Typhimurium can occur via distinct pathways (overexpression and mutagenesis of fab1; active efflux via AcrAB–TolC), and that mutants selected after a single exposure to triclosan are fit enough to compete with wild-type strains. Interestingly, within bacterial biofilms, triclosan also up-regulated the transcription of acrAB, a gene encoding for the main efflux pump in Gram negative bacteria, of marA, the major regulator of the genetic cascade controlling multi-drug resistance and of the cellulose-synthesis coding genes bcsA and bcsE. Therefore, when present within biofilms, Salmonella can drastically alter its membrane permeability via decrease of porin synthesis, increased efflux and enhanced exopolysaccharides production (Tabak et al. 2007). This alteration of membrane permeability may induce a serious decrease of the susceptibility to various antimicrobial molecules including biocides and antibiotics.

In some circumstances, a specific mechanism has not been established and a phenotypic change leading to the emergence of resistance to several unrelated compounds in vitro has been reported following exposure to a low concentration of a biocide (Chapman 2003, Thomas et al. 2005, Walsh et al. 2003). The treatment of E. coli with PHMB induced the alteration of transcriptional activity in a number of genes, notably in the rhs gene involved in repair/binding of nucleic acid (Allen et al. 2006). Exposure to an oxidising biocide produced an alteration of protein expression in resistant S. enterica mutants consistent with the production of a stress response and in particular the expression of detoxifying enzyme. Exposure to phenol-based disinfectant also produced a change in protein expression consistent with the expression of an efflux pump system (Randall et al. 2007).

Quorum sensing might also have a role in the establishment of a resistant phenotype (Davies et al. 1998, Hassett et al. 1999, Shih and Huang 2002), although this might be biocide specific. MacLehose et al. (2004) provided evidence that homoserine lactone (HSL) mediated quorum sensing was not involved in Ps aeruginosa biofilm susceptibility to QAC and chlorhexidine, but could be involved with bronopol. Further evidence of the role of quorum sensing in the development of resistance is necessary (MacLehose et al. 2004)

Source & ©: SCENHIR,  Assessment of the Antibiotic Resistance Effects of Biocides (2009),
Section 3.4.3.Mechanisms of resistance to biocides, p. 30-34


4.2 How can bacteria become resistant to antibiotics?

The SCENIHR opinion states:

3.5. Bacterial resistance mechanisms

3.5.1. Resistance mechanisms to antibiotics

Resistance to antibiotics may result from innate (intrinsic) or acquired mechanisms.

Intrinsic resistance is a trait of a bacterial species. For example, the target of the antimicrobial agent may be absent in that species, the cell envelope (cell membranes and peptidoglycan) may have poor permeability for certain types of molecules or the bacterial species may produce enzymes that destroy the antimicrobial agent. These bacteria are clinically resistant, but should more accurately be referred to as “unsusceptible”, as it is often merely a matter of increasing the concentrations of the antimicrobial agent to levels that may never be reached during therapy, or only at certain sites.

A bacterial strain can acquire resistance either by mutation or by the uptake of exogenous genes by horizontal transfer from other bacterial strains. Genes encoding enzymes that can modify the structure of an antimicrobial are commonly transferable (penicillinases and cephalosporinases (bla-genes), acetyl transferases modifying e.g. aminoglycosides (aac-genes), as are genes leading to target modification (erm-genes), methicillin-resistance (mecA-genes) and glycopeptide-resistance (van-genes). There are several mechanisms for horizontal gene transfer, mainly based on mobile genetic elements, which often function in concert (Dobrindt 2004). Large plasmids with many different genes can be transferred from bacterium to bacterium by conjugation. Transposons can carry several resistance genes. They cannot replicate by themselves, but can move within the genome, e.g. from plasmid to plasmid or from chromosome to plasmid. Integrons can also encode several resistance genes. They cannot move by themselves, but encode mechanisms both to capture new genes and to excise and move cassettes with genes within and from the integron. Integrons are commonly carried on plasmids (EFSA, 2005), but may also be chromosomally-integrated such as in Salmonella Typhimurium DT 104. Antibiotics, targets and activities

The diverse antibiotic molecules used during antibiotherapy of bacterial infections may be classified according to their mechanism of action on bacterial cell. There are 4 major mechanisms: (1) alteration of cell envelope, (2) inhibition of protein synthesis, (3) inhibition with nucleic acid synthesis, and (4) inhibition of a metabolic pathway (see Table 10 ).

The ß-lactams (penicillins, cephalosporins, carbapenems, etc), polymyxins, CAMPs and glycopeptides (vancomycin and teicoplanin) work by perturbing the bacterial cell wall synthesis or the membrane stability/integrity. ß-lactam molecules block synthesis of the bacterial cell wall by interfering with the enzyme activity involved in the final step of peptidoglycan synthesis. Polymyxins and cationic antimicrobial peptides exert their inhibitory effects by increasing bacterial membrane permeability, causing leakage of bacterial contents (ions, ATP etc.). The cyclic lipopeptide daptomycin induces depolarisation of the outer membrane and subsequent cell death by inserting its lipid part into bacterial membrane. Vancomycin and teicoplanin interfere with the final cross-linking steps of pentapeptide units during cell wall synthesis preventing stable cell wall synthesis.

Table 10: Mechanisms of action of antibiotics 

Macrolides, aminoglycosides, tetracyclines, chloramphenicol, streptogramins and oxazolidinones inhibit various steps involved in protein synthesis: macrolides, aminoglycosides, and tetracyclines bind to the subunits of the ribosome or to rRNA (e.g. S12 protein, 23S rRNA etc.), whereas chloramphenicol binds to the 50S subunit interfering with the translation process.

Fluoroquinolones exert their antibacterial effects by disrupting DNA synthesis and causing lethal double-strand DNA breaks during DNA replication (inhibition of gyrase and topoisomerase activities) whereas sulfonamides and trimethoprim block the pathway for folic acid synthesis, which ultimately inhibits DNA synthesis. The drug combination of TMP, a folic acid analogue, plus sulfamethoxazole (a sulfonamide) inhibits steps in the enzymatic pathway for bacterial folate synthesis. Main bacterial mechanisms of antibiotic resistance

Bacteria may resist antibiotic action by using several mechanisms. Some bacterial species are innately resistant to one class of antibiotics, e.g. bacteria are resistant due to their intrinsic envelope that limits the antibiotic penetration or to the presence of a low level of efflux systems that decrease intracellular antibiotic concentration (Nikaido, 2003; Li and Nikaido, 2004). In such cases, all strains of that bacterial species are likewise resistant to all the members of those antibacterial classes (see Definition section

An ongoing and increasing concern is bacteria that become resistant: e.g. initially susceptible bacteria become resistant to antibiotics and consequently disseminate under the selective pressure of use of these antibiotics (which kill other competitive bacteria). Several mechanisms of antimicrobial resistance are readily spread to a variety of bacterial genera.

A simple technical definition of the various resistance mechanisms may be proposed for classification: mechanical barrier (altering the required intracellular dose of antibiotic); enzymatic barrier (expression of a detoxifying enzyme that modifymodifies the antibiotic); target protection barrier (mutation or expression of a molecule impairing the antibiotic recognition and activity) (see Table 11 ).

Table 11: Major resistance mechanisms (Davin-Regli et al., 2008) 

Mechanical barrier mechanism

  • Bacteria may modify membrane permeability, such as a decrease of porin content or an alteration of the LPS structure, two responses that prevent the antibiotic access to the target at required concentrations (minimal inhibitory concentration).
  • Alternatively or conjointly, bacteria may produce efflux pumps that extrude the antibacterial agent from the cell before it can reach its target site and exert its effect.

Enzymatic barrier mechanism

  • Bacteria may acquire plasmid genes or over-expressed chromosomal genes encoding enzymes that cleave the antibacterial agent before it can have an effect, such as ß-lactamases, cephalosporinases etc.
  • Bacteria may acquire several genes for other modifications of the antibiotic such as acetyltransferase, phosphotransferase etc.

Target protection barrier mechanism

  • Bacteria may protect the antibiotic target by acquiring mutations that strongly decrease the affinity of the antibiotic for the target, by producing mimicked targets that lure antibiotics.
  • Bacteria may synthesise a protective molecule masking the target access to antibiotics.

Consequently, susceptible bacteria may exhibit an efficient level of resistance to antibiotics via mutation and selection, by expressing special resistance mechanisms (down-regulation of porins, overproduction of efflux pumps etc.) in response to external stimuli, or by acquiring from other bacteria the genetic information that provides resistance mechanism (e.g. gene for enzyme, efflux transporter). The last event may occur by several genetic mechanisms including transformation, conjugation or transduction. Multi-drug resistant bacteria

Many bacteria have become resistant to multiple classes of antibiotics (at least three unrelated antibiotic classes) and deploy multiple strategies to overcome the stress of antibiotic chemotherapy. Resistance is not necessarily limited to a single class of antibiotics. It can apply, simultaneously, to many chemically unrelated compounds to which the cell has never been exposed: this is termed « multi-drug resistance » (MDR).

Today, these MDR bacteria are a cause for serious concern in hospitals and other health care institutions where they are commonly detected. The major mechanism of MDR is the active transport of drugs from the cell to the environment by pumps which expel a broad spectrum of compounds that are noxious to the bacterium (including antibiotics, biocides etc.). In addition, the poly-specificity of efflux transporters confers a general resistance phenotype that can reinforce the effect, and/or drive the acquisition of additional mechanisms of resistance such as mutation of antibiotic targets or synthesis of enzymes that alter the drugs.

There is strong evidence for the role of AcrAB-TolC efflux in Enterobacteriaceae: the expression of this efflux pump is an important prerequisite for the selection of fluoroquinolone resistant mutants that exhibit mutated targets (mutation in gyrase and topoisomerase) in various Gram-negative bacteria such as Salmonella or Campylobacter, two major food-borne pathogens (Piddock 2006). These two mechanisms, conjointly expressed, confer a high resistance level against quinolones. Similar synergies have been recently reported for macrolides in Campylobacter and other examples may be mentioned with ß-lactams, CAMPs, polymyxins, and Enterobactericeae (Davin-Regli et al. 2008, Piddock 2006).

In all of these cases, strains of bacteria carrying resistance factors are selected by the use of antimicrobial molecules which kill the susceptible strains but allow the newly resistant strains to survive and grow. Acquired resistance due to chromosomal mutation and selection is termed vertical evolution since the advantage will be conferred to a bacterial line. Bacteria also develop resistance through the acquisition of new genetic material from other resistant organisms. This is termed horizontal transfer, and may occur between strains of the same species or between different bacterial species or genera sharing a same ecological niche. Mechanisms of genetic exchange include conjugation, transduction, and transformation. For each of these processes, transposons facilitate the transfer and incorporation of the new resistance genes into the genome of the bacterial host or into plasmids

Source & ©: SCENHIR,  Assessment of the Antibiotic Resistance Effects of Biocides (2009),
Section 3.5.1Resistance mechanisms to antibiotics, p. 39 - 42


4.3 Which resistance mechanisms are common to both biocides and antibiotics?

The SCENIHR opinion states:

3.5.2. Common resistance mechanisms

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 2004b, Gilbert and McBain 2003, McBain et al. 2002, McBain et al. 2003, Pumbwe et al. 2007, Russell 2004a and b, 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 the number of studies in the clinical or environmental setting is low. However, a recent study performed in the community highlighted a significant relationship between high QAC MICs, high MICs to triclosan and resistance to one or more antibiotics (Carson et al. 2008).

Further research is needed to establish a correlation between biocide exposure(s) and development of antibiotic resistance. Biocides tend to act concurrently on multiple sites within the microorganism, and thus resistance is often mediated by non-specific means. 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 (Maillard 2007, Poole 2007,). 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 for antibiotic resistance has also been described (Fraise 2002). Biocides and antibiotics share common resistance mechanisms

Several publications and reviews have presented the cell target of biocides and the various mechanisms used by the bacterial cell to evade the toxic activity of biocides (for recent reviews see Denyer and Maillard 2002, Gilbert and Moore 2005, Lambert 2002, Lambert 2004, Maillard 2002, Maillard 2007, Poole 2004, Stickler 2004). It is important to note that antibiotic and biocide antibacterial actions show many similarities despite some differences in terms of target, killing, behaviour and clinical aspects (Poole 2007). Among the similarities, we can mention (i) the penetration/uptake through bacterial envelope by passive diffusion, (ii) the effect on the membrane integrity and morphology, (iii) the effect on diverse key steps of bacterial metabolism (replication, transcription, translation, transport, various enzymes). Faced with this toxic effect and stress, the response/adaptation of bacterial cells presents some similar defence mechanisms that can overlap the original functions to confer resistance against structurally non-related molecules. Among the biocide resistance strains intrinsic and acquired mechanisms are described (see section 3.1.3).

Intrinsic resistance is an innate property conferred by the bacterial genome (species-dependant) and includes impermeability, efflux, biofilms and transformation of toxic compounds. To decrease the intracellular concentration of noxious molecules, Gram-negative bacteria can regulate the permeability of their membranes by decreasing the synthesis of porins (membrane pore-forming proteins involved in antibiotic uptake) and modifying the lipopolysaccharide structure (Nikaido 2003, Poole et al. 2002a) or overexpressing the efflux pumps (membrane proteinous complexes involved in antibiotic expulsion) (Poole 2007). These strategies are involved in the resistance against antibiotics and biocides (Thorrold et al. 2007). In parallel, the acquired resistance occurs via mutation and acquisition of mobile DNA (transposon, plasmids) coding for resistant elements (enzyme, transporter).

Similarly, the acquired processes may protect against antibiotics and biocides (Maillard 2007). In addition, some of the mechanisms that play a major role in resistance are controlled by diverse genetic cascade regulations that share common gene regulators (soxS, marA) (Poole 2007). Bacterial biofilms and resistance

In practice, most bacteria are associated with surfaces and grow as biofilm rather than as planktonic cells. Bacterial biofilms have been consistently described as being more resistant to biocides and antibiotics than planktonic cells (Bisset et al. 2006, Gilbert et al. 2003, Maira-Litrán et al. 2000, Smith and Hunter 2008). The reasons for this decrease in susceptibility is a biofilm-associated phenotype (Ashby et al. 1994, Brown and Gilbert 1993, Das et al. 1998), including decreased metabolism, quiescence, reduced penetration due to the extracellular polymeric matrix (Pan et al. 2006), enzymatic inactivation of biocides (Giwercman et al. 1991, Huang et al. 1995, Sondossi et al. 1985), and the induction of multi-drug resistant operons and efflux pumps (Maira-Litrán et al. 2000).

Although bacteria within biofilms are undeniably more resistant to biocides and antibiotics, the link between the uses of biocides against bacterial biofilm and potential emerging antibiotic resistance is not straightforward. In a recent study investigating the use of chloraminated drinking water against Ps. aeruginosa biofilm, there was no evidence that the use of chloramine induced an increase in antibiotic resistance (Jurgens et al. 2008). Induction of antibiotic resistance by biocide molecules

A key question is whether the use of biocides facilitates the selection of antibiotic resistant bacteria. It is quite difficult to obtain a clear response considering that (i) the only available data focus on specific molecules or specific bacteria and (ii) there is always a difference between the in vitro and in vivo analyses. However, some published data concerning the relationships between antibiotic resistance and biocide resistance can be mentioned.

Recent studies carried out on two important pathogens, Salmonella enterica and Stenotrophomonas maltophila described the effect of the bisphenol triclosan on emerging bacterial cross-resistance. In the first work concerning Salmonella, the authors reported that triclosan-selected strains are less susceptible to antibiotics than the wild type original strain (Karatzas et al. 2007). The overexpression of an efflux pump (SmeDEF), involved in antibiotic resistance, is demonstrated in the various triclosan-selected clones (Sánchez et al. 2005). A more recent study described the survival of S. enterica serovar Typhymurium following exposure to various disinfectants at a low concentration on the resulting changes in antibiotic profile (Randall et al. 2007). They concluded that growth of Salmonella with sub-inhibitory concentrations of biocides favours the emergence of strains resistant to different classes of antibiotics. In Stenotrophomonas, the authors analysed the effect of triclosan and phenolic farm disinfectants on the selection of antibiotic derivative strains (Sánchez et al. 2005). Other investigations described Pseudomonas aeruginosa overexpressing multi-drug efflux systems during exposure to chlorhexidine (Fraud et al. 2008). In the same way, the exposure of clinical isolates of Staphylococcus aureus results in the selection of strains which over-express several resistance genes (Huet et al. 2008).

Similar results have been reported with S. enterica and Escherichia coli (Braoudaki and Hilton 2004a). E. coli O157 strains, involved in the hamburger disease, acquired high- levels of resistance to triclosan after only two sublethal exposures and when adapted, repeatedly demonstrated decreased susceptibilities to various antibiotics, including chloramphenicol, erythromycin, imipenem, tetracycline, and trimethoprim, as well as to a number of biocides. These observations raise concerns over the indiscriminate and often inappropriate use of biocides, especially triclosan, in situations where they are unnecessary, whereby they may highlight their potential role in contributing to the development of microbial resistance mechanisms. Moreover, a well-conducted study demonstrated that biocide (i.e. polyquaternium-1) and antibiotic resistance mechanisms were linked at the genetic level (Codling et al. 2004). A transcriptional study has demonstrated that paraquat is able to induce the expression of several genes involved in antibiotic resistance (Pomposiello et al. 2001). Regulation pathway and overlap between biocides and antibiotics: the sox regulon

In E. coli, and S. enterica, mar and sox regulons play a key role for the induction of multi-drug resistance (Levy 2002, Poole 2007). The soxS protein is the direct activator of genes for resistance to both oxidants and antibiotics. In laboratory strains of E. coli and S. enterica, activation of the soxRS regulon with paraquat treatment increased resistance to ampicillin, nalidixic acid, chloramphenicol, and tetracycline. Moreover, the soxRS regulon was also connected to antibiotic resistance in clinical strains (Koutsolioutsou et al. 2005). Constitutive soxS expression contributed significantly to the quinolone resistance of an S. enterica clinical isolate, caused by a soxR mutation (repressor of sox regulon) that evidently arose during clinical treatment.

Sixteen per cent of fluoroquinolone-resistant, organic solvent-resistant clinical E. coli isolates exhibited constitutive soxS expression. Twenty-eight per cent of fluoroquinolone-resistant clinical and veterinary E. coli isolates exhibited constitutively elevated soxS expression. This moderate, multiple-antibiotic resistance is a hallmark of soxRS-mediated mechanisms that are involved in biocide and antibiotic resistance. This overlap is of interest when a bacterial strain (potential nosocomial pathogen) is exposed to biocides

Source & ©: SCENHIR,  Assessment of the Antibiotic Resistance Effects of Biocides (2009),
Section 3.5.2. Common resistance mechanisms, p. 42- 44

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