8. How can risks of resistance to both antibiotics and biocides be assessed?
- 8.1 What factors increase the risk of resistance to both biocides and antibiotics?
- 8.2 What (new) methods are required to effectively assess the risk of resistance?
The SCENIHR opinion states:
3.10. Risk assessment
The selection of resistant or insusceptible bacteria following exposure to a biocide should be considered. A number of studies highlighted selection for resistant bacteria clones although the antibiotic phenotype was not necessarily determined. Several laboratory scale investigations demonstrated the selection for bacteria showing an increased tolerance to a biocide following treatment with a low concentration of a biocide (Abdel Malek et al. 2002, Langsrud et al. 2003, Tattawasart et al. 1999, Thomas et al. 2000, Walsh et al. 2003). Gaze et al. (2005) reported QAC selection in the natural environment following QAC exposure. Recently the effect of triclosan in selecting for small colony variants of S. aureus was described, highlighting a potential detrimental effect for strain identification and subsequent miss-diagnosis in a clinical context (Seaman et al. 2007).
Antibiotic use is still the major cause of antibiotic resistance in clinical practice. Since antibiotic resistance remains a major concern and decreases our ability to treat infections, appropriate infection control strategies are paramount and involve prevention through good hygiene which encompass the appropriate use of biocides (OJEC 1999, Department of Health 2000).
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Section 3.10. Risk assessment, p.55
8.1 What factors increase the risk of resistance to both biocides and antibiotics?
The SCENIHR opinion states:
3.10.1. Categorisation of potential factors involved in the biological risk
188.8.131.52. Predisposition of bacterial species to acquire resistance
Horizontal gene transfer, a fundamental mechanism for the evolution of microbial genomes, is the main cause of dissemination of resistance determinants. This non-parental transfer of genetic material from one organism to another, such as from one bacterium to another or from viruses to bacteria, is pervasive and plays a relevant role in accelerating the spread of antibiotic resistance (for details see sections 3.4/3.5/3.9). The three mechanisms of horizontal gene transfer identified are transduction, transformation and conjugation.
Transduction involves the accidental packaging of cellular DNA into bacteriophage particles during replication. Transformation is the uptake of free DNA by a bacterial cell and its stable integration into the bacterial genome, but bacterial conjugation is the most efficient system of horizontal gene transfer in bacteria.
In this process, DNA is transferred from donor to recipient bacteria by specialised machinery: the conjugation apparatus, which includes molecular mechanisms responsible for intimate cell-cell contact and for the transfer of mobile genetic elements. As components of the horizontal gene pool, mobile genetic elements include insertion sequences, transposons, integrons, bacteriophages, genomic islands (such as pathogenicity islands), plasmids and combinations of these elements.
Although mechanisms of gene transfer occur in both bacteria and archaea, some bacterial groups seem to have developed highly efficient mechanisms for gene transfer. While gaps in information make it difficult to categorise bacterial species according to their efficiency in conjugative gene transfer, the available scientific information still allows the definition of three categories related to this potential risk:
a. High: bacterial species for which highly specialised mechanisms for high frequency gene transfer have been described (e.g. Enterococcus Enterobacteriaceae); high probability of exchange between unrelated species or to virulent strains.
184.108.40.206. Induction of antibiotic resistance gene via genetic cascade
Several genetic cascades control the induction of the expression of general/non-specific resistance mechanisms including efflux pumps and permeability change. Among them, genetic activators such as SosS or MarA can be activated by several chemicals such as biocide molecules (Blanchard et al. 2007, Davin-Regli et al. 2008, Pomposiello et al, 2001). Taking into account this chemical activation, biocides may induce the expression of antibiotic resistance cascades in susceptible strains generating a decrease of antibiotic susceptibility, or, select bacteria which express the corresponding genes.
In addition, in integrative elements such as transposons, plasmids etc. several genes involved in biocide and antibiotic resistance co-segregate (Dobrindt 2004; Gaillard 2008). This genetic linkage favors the selection and the dissemination of resistant bacteria carrying these mobiles elements. Moreover, the transfer of such key genes will be increased under selective pressure such as the presence of biocides.
220.127.116.11. Type of antimicrobial (intrinsic potential for generating resistance)
Based on our current state of knowledge and literature based evidence from mainly in vitro studies, bacteria have been shown to be able to withstand biocide exposure. The mechanisms by which bacteria can escape damage from biocides are complex and multiple, and are governed by a number of factors inherent to the biocide (e.g. concentration, contact time etc.) and to the bacteria (e.g. type, metabolic activity).
However, some biocides, because of the nature of their interaction with the bacteria, would be more prone to induce resistance/tolerance. This group of high-risk biocides contains the quaternary ammonium compounds, biguanides (i.e. surface active agents) and phenolics. Metallic salts, such as silver could also be added to this list based on practice-based evidence from the 1960s-1970s.
Highly reactive biocides such as oxidising agents and alkylating agents would present a low risk when emerging bacterial resistance is concerned. This means that resistance is unlikely but not impossible. Examples of resistance to these biocides have been described, but they resulted mainly from an inappropriate usage of the biocide.
Finally, for a number of biocides used heavily in consumer products and in the food industry (e.g. isothiazolones, anilides, diamidines, inorganic acids and their esters, alcohols), there is little information available on emerging resistance/tolerance when bacteria are exposed to their in-use concentrations. However, because of the nature of their interaction with the bacterial cell and their antimicrobial efficacy, these biocides would have to be classified for the time being as being of a medium risk in terms of emerging bacterial resistance until they can be properly assessed.
This point is very difficult to evaluate due to the missing data for the tonnages used and the distribution of the many molecules.
18.104.22.168. Form of growth
Bacteria are able to grow either free in media (planktonic status) or as part of a sessile community, forming biofilms. This represents a protected mode of growth that allows cells to survive in hostile environments (see section 3.7.3).
The presence of conditions which allow the formation of bacterial biofilm could be considered as a potential risk for the development of cross-resistance between antibiotics and biocides. Examples are:
- Prosthetic materials, implants, catheters;
- Food and chemical plants;
- Water and wastewater treatment plants (filters, flocks, trickling filters).
22.214.171.124. Environmental factors
The environmental factors which may play a role in the bacterial response (adaptation) may include: the type of bacterial community, temperature, oxygen level, nutrient levels, pH of the medium, detergents, exposure time etc. All these factors may influence the growth, the metabolism/physiology of the bacterial cell and the division cycle which are key points in the bacterial susceptibility. In addition, they also are involved in the transfer of genetic elements, the quorum sensing (transduction of cell-cell signal) and the formation of biofilm (see above).
126.96.36.199. Prevalence of bacterial species
This important point is related to:
- The biological aspects, including the bacterial species submitted to selective pressure, involved in the transmission of MGE and directly involved in the biological hazard (final host) (see example of biological hazard 3.9.1-2 );
- The respective concentration of biocides (as active stress agent) and the time of contact (point 4);
- The types of biocides present in the bacterial environment and their chemical properties (stability, affinity for bacterial target, bioavailability etc.).
3.10.2. Risk factors for resistance antimicrobials
The large and indiscriminate use of (biocidal) chemical compounds will increase the wide dissemination of mobile genetic elements (see section 3.9.1). This is already often the case, for example in agriculture, breeding, intensive farming and rearing. As biocidal substances are used in numerous domestic and industrial products and applications, they come in direct contact with the soil and the associated microflora via wastes, faeces etc. Soil is a general reservoir of many environmental bacteria and opportunistic pathogens (Gram-negative and -positive bacteria) containing a large diversity of mobile genetic elements that contain resistance genes.
Among this microflora several Gram-negative and -positive bacteria may be (i) selected because the genes involved in biocide resistance are actively present on the chromosome (genetic island) or on mobile elements (plasmids), or (ii) because the bacteria is able to acquire corresponding mobile genetic elements from the neighbouring bacteria. Under the presence of biocides, and due to the presence of resistance gene targeting both antibiotics and biocides on mobiles genetic elements, genetic rearrangements are favoured inducing the intra and inter-species dissemination of such key genes.
This risk concerns not only the soil bacteria but also the bacteria that colonize the various farm animals (Campylobacter, Enterococcus, Salmonella etc.) which are in contact with environmental bacteria (Pseudomonas etc.) containing these mobile genetic elements. Consequently, the risk can spread to food-borne pathogens which are frequently detected in animals. For example, the dissemination of resistance genes may affect Campylobacter, Escherichia, Salmonella and several mobile genetic elements containg biocide and antibiotic resistance genes have been described (see section 3.7.2).
The dissemination of mobile genetic elements conferring the resistance against biocide-antibiotic is clearly evidenced, the possibility that this event concerns important food-borne pathogens is also reported, the human exposure to this event is also important (via food-borne pathogens or nosocomial infections).
To conclude, the hazard exists for several human pathogens and this may concern a significant part of the population
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Section 3.10.1 Categorisation of potential factors involved in the biological, p.55 - 58
8.2 What (new) methods are required to effectively assess the risk of resistance?
The SCENIHR opinion states:
3.10.3. Requirements for new methodologies for risk assessment of the effect of biocide usage on antibiotic resistance
Protocols for testing the antimicrobial efficacy of biocides are essential to provide reliable information on the efficacy of an antimicrobial product and provide assurance for the end users. Variability in results observed in the literature often resides in the differences in protocols used, some tests being less stringent than others (Kampf et al. 2003; Marchetti et al. 2003, Messager et al. 2004), but also the non-respect of test preparation (notably inoculum) and conditions (Jacquet and Reynaud 1994, Taylor et al. 1999).
There are no internationally agreed standard protocols and often countries have their own government laboratory testing with their own standards, although in Europe, CEN/TC 216 (the European Committee for Standardisation) aims to produce current and future European disinfectant testing standards (Holah 2003). Test methodology can range from basic preliminary suspension tests to more complex protocols that simulate conditions in practice. The purpose of antimicrobial efficacy testing is to determine a pass/fail criterion for a given biocide under specific conditions. The design of efficacy test protocols for biocides is complex notably because of the number of factors that need to be controlled. These factors can be divided into those depending upon the micro-organism (e.g. test strain, preparation of inocula, detection and count of survivors) and those depending upon the test method (e.g. quenching antimicrobial activity, physical parameters). There are a number of protocols available for testing the antimicrobial efficacy of biocides (Lambert 2004).
One of the major limitations of efficacy test protocols is their reproducibility and robustness (Bloomfield and Looney 1992, Bloomfield et al. 1994, Bloomfield et al. 1994, Borgmann-Strahsen 2003, Kampf and Ostermeyer 2002, Kneale 2003, Langsrud and Sundheim 1998, Tilt and Hamilton 1999). In addition, practical tests conducted in laboratory conditions that aimed to simulate conditions in the field, might sometimes be too rigid and do not allow much flexibility which impinge on the ability to set parameters reflecting conditions found in practice. On the other hand tests in loco are costly and difficult to standardise since parameters cannot be controlled accurately in the field. These tests remain poorly reproducible and their outcomes might be contentious, although they would provide key information on the antimicrobial efficacy of biocides to the manufacturers and end users.
There are no standardised testing protocols that measure both biocide and antibiotic resistance in bacteria. Often environmental and clinical isolates have been tested for their susceptibility to biocides and antibiotics in separate efficacy test protocols. Undoubtedly, the use of a range of diverse protocols, some based on MIC determination (as discussed previously), adds to the variability in information in the literature. Thus there is an urgent need for the design of a standardised test to determine both biocide and antibiotic resistance in bacterial isolates.
In addition, the role of bacterial biofilm in resistance to both biocides and antibiotic has been shown. Furthermore, bacterial biofilms have been deemed to provide a better representation of how bacteria are present in the environment. However, most laboratories are not using biofilm tests to assess the efficacy of biocides (Cookson 2005). There are currently no European standards for the testing of disinfectants against biofilms for health care applications. This is particulalrly pertinent since there is evidence that the complete elimination of a biofilm is difficult and might not happened even where stringent cleaning procedure are in place (Pajkos et al. 2004).
However, since bacteria (and notably environmental/clinical isolates) grown as a biofilm are more resilient to antimicrobial action (Kimiran-Erdem et al. 2007), one of the problems associated with biofilm efficacy test, apart the type of protocol to be used, is that higher a concentration of a biocide will most probably have to be used to ensure efficacy. This will lead to increase in costs for the manufacturer and increase in levels of biocide released in the environment
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Section 3.10.3 Requirements for new methodologies for risk assessment of the effect of biocide usage on antibiotic resistance, p.58 - 59
3.10.4. Quantitative approach
A) Specific use situations
The preceding discussion indicates that, on mechanistic grounds, it is reasonable to assume that under certain circumstances, frequent exposure to minimum selective concentrations will trigger antibiotic resistance.
The likelihood of this occurring and its relative importance will depend on:
- How the biocide is used i.e. the exposure conditions (type of surface, concentration in use etc.).
- The microbial pathogens exposed.
- Environmental factors that may favour the selection of resistant pathogens.
- Exposure time.
Assessment of exposure is inevitably specific to usage. Key parameters are the duration of exposure and remaining concentration. Two particular situations need particular consideration:
- Frequent release/application of one or more biocides that enable non-lethal concentrations or sub-inhibitory concentration to be maintained in pathogen rich locations.
- Biocides that are environmentally persistent and can maintain a residual concentration below the minimum inhibitory concentration because they can maintain selective pressure.
i) Microbial pathogens
The relative susceptibility of bacteria is the consequence of intrinsic and acquired resistance mechanisms (see sections 3.4 and 3.5). It is now clear that intrinsic resistance is an evolutionary advantage for bacteria: it has evolved to maintain a minimal protection against harmfull compounds and is genetically conserved (vertical transmission). For instance, low permeability of the bacterial envelope or efficient polyselective efflux pumps allows bacterial cells to survive harmful chemical and physicial stresses. The level of un-susceptibility depends on the bacterial genera and sometimes species, and can increase with (over)expression of specific genes following exposure to environmental factors and specific stresses (toxic agents etc.). In addition, the presence of overlapping cascades of regulation controlling resistance genes may increase the resistance level. The acquisition of new resistant determinants (acquired resistance; horizontal transfer) may be beneficial to the bacteria under specific stressful conditions, but may have an environmental cost when no selective pressure is present.
ii) Other environmental factors that may influence resistance
All factors acting on bacterial physiology (see section 188.8.131.52) can modulate the level of bacterial susceptibility and trigger or favor the selection or emergence of resistant strains. For instance, oxygen may de-repress the Sox operon which is a part of the regulation cascade inducing the expression of the efflux mechanism; pH and divalent cations may induce some changes in the envelope structure (e.g. proteins, lipopolysaccharide) decreasing the penetration of antibacterial molecules. All environmental factors (chemical, physical, biological etc.) which alter the normal permeability of the envelope are likely to promote a change in susceptibility.
It is important to consider the relative contribution of the use of a particular biocide compared with that of antibiotics. In situations where there is extensive use of antibiotics this exposure plays inevitably a dominant role in emerging antibiotic resistance. However, the use of biocides in such settings (e.g. hospitals) may also contribute to the selection of bacterial genera and species that are less susceptible to the biocide used and show cross-resistance to certain antibiotics.
In other situations such as food manufacturing there may be extensive use of biocides with minimal or no use of antibiotics. Consequently, it is appropriate to consider the risk from biocide exposure in the emergence or development of resistant bacterial strains.
B) Assessment of the generic risk
Assessment of the generic risk within the European Union requires information on:
- The current and likely future uses of biocides in the EU. This includes the tonnage of particular biocides in current use. Regrettably, the industry has been unwilling to provide this information and hence any assessment of the generic risk is impossible at present.
- The minimum selective concentrations of each of these biocides
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Section 3.10.4 Quantitative approach, p. 59 - 60