7. What explains resistance to both biocides and antibiotics?
- 7.1 How can biocide exposure lead to antibiotic resistance?
- 7.2 In practice, does resistance emerge in homes and the environment?
- 7.3 How can dissemination of resistance genes lead to resistance?
- 7.4 How can the formation of biofilms lead to resistance?
7.1 How can biocide exposure lead to antibiotic resistance?
The SCENIHR opinion states:
3.8.5. Relationship between biocide resistance and antibiotic resistance
- Cross-resistance: selection for genes encoding resistance to both the biocidal substance and one or more therapeutic antibiotic classes. The term 'cross-resistant' is used to denote a strain possessing a resistance mechanism that enables it to survive the effects of several antimicrobial molecules.
- Change in the physiological response of the bacterium following biocide exposure, resulting in a decrease in susceptibility to both biocidal substance and antibiotics.
- Co-resistance: selection for clones or mobile elements also carrying antimicrobial resistance. Co-resistance refers to genetic determinants conferring resistance present on the same extrachromosomal element, transferred and expressed jointly in a new bacterial host.
- Indirect selection for bacterial sub-population following biocide exposure resulting in a decrease in susceptibility to both biocidal substance and antibiotics.
- Enhanced DNA repair e.g. by activating a SOS response in bacteria (SOS response is an inducible DNA repair system that allows bacteria to survive sudden increases in DNA damage.).
Unfortunately there is no complete report in the literature reporting at the same time on all five principles. Instead researchers have usually limited their investigations to one or two principles, potentially missing some important information on linkage between biocide and antibiotic resistance.
In antibiotics, cross-resistance has been described well. Antibiotics are a diverse group of molecules, commonly ordered in classes with similar structures and modes of action. Within a class, the target in the bacterial cell and the mode of action of the antibiotics is the same or similar. Therefore, some mechanisms of resistance will confer resistance to most or all members of a class, i.e. cross-resistance. Cross-resistance may also occur in relation to unrelated classes, if the target overlaps (as in the case of macrolides and lincosamides) or if the mechanism of resistance is of low specificity.
In very few instances cross-resistance between biocides and antibiotics has been described. Such resistance involved mainly efflux pumps mediating reduced susceptibility to both classes of antimicrobial agents (Levy 2002, Piddock 2006, Thorrold et al. 2007). However, in other instances changes in cell envelope (reduction in porins and changes in LPS and other lipids) has been described (Denyer and Maillard 2002, Nikaido 2003, Tkachenko et al. 2007). Finally, the role of bacterial biofilm in conferring resistance to both antibiotics and biocides cannot be ignored.
Co-resistance can occur when mechanisms encoding resistance or reduced susceptibility are genetically linked. Genes conferring antimicrobial resistance are frequently contained in larger genetic elements such as integrons, transposons or plasmids, and as such may be ‘linked’ to other, unrelated resistance genes. In such cases, multiple resistance genes may be transferred in a single event. Consequently, selection for one resistance gene will also select for the other resistance gene(s). For example, this is the case for tolerance to quaternary ammonium compounds in Gram-negative bacteria. The qac-genes are often together with sul1 genes encoding sulphonamide resistance located as part of mobile genetic elements which also can harbour various other resistance genes (Sidhu et al. 2001, Sidhu et al. 2002). Resistance genes can be located on mobile genetic elements or in the bacterial chromosome. Co-resistance has also been described in Salmonella enterica with metallic salts such as organomercurials (Levings 2007). Exposure to a biocide causes major stress. Thus, it must be expected that a biocide can initiate a SOS response in a bacterium, promoting horizontal gene transfer of resistance genes (Beaber et al. 2004, Ubeda et al. 2005).
In laboratory settings, the use of biocides has been shown to select indirectly for resistance to antibiotics by causing a clonal drift in the bacterial population towards bacterial cells that are more resistant. As an example the emergence of multi-drug resistant Salmonella enterica serovar Typhimurium DT104 caused an overall increase in the occurrence of resistance to antibiotics among Salmonella from food animals and humans in several countries (Doublet et al. 2003, Doublet et al. 2008)
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Section 3.8.5. Relationship between biocide resistance and antibiotic resistance, pg. 51 - 52
7.2 In practice, does resistance emerge in homes and the environment?
The SCENIHR opinion states:
3.8.6. Tonnages and exposure
To assess the general exposure of human and the environment, knowledge about production and uses of various biocides is required. However, information concerning production and use of biocides in the open literature is sparse. The WG attempted to get such information by publishing a Call for Information on an EU Website, and by contacting various DGs within the European Commission as well as relevant Member State Authorities. No useful information in this respect was obtained from any side. In the absence of adequate knowledge on product and use of biocides, an alternative strategy for exposure assessments was required.
A practical approach may be based upon the exposure concentrations and frequency of exposure, considering the aggregate exposure when relevant. In the case of the bacterial flora in the home environment, repeated exposures to biocides in cleaning products, disinfection products and other relevant products could be considered to be a continuous selective pressure allowing the potential emergence of well-adapted strains.
Environmental concentrations of many biocides in air, water and soil are reported in open literature and various databases. A continuous exposure of bacterial flora by biocides in natural environments should be considered for the estimation of development of antibiotic resistance.
3.8.7. Appearance of resistance in practice
It is clear from in vitro studies that bacterial resistance can develop rapidly following exposure to a biocide. The initial stress response caused by a biocide, which does not demonstrate a lethal action, is rapid and has been exemplified by the initiation of a SOS response or has been indirectly demonstrated by looking at growth curve in the presence of a biocide (Gomez-Escalada et al. 2005a). It is difficult to ascertain how wide spread the development of bacterial resistance to a biocide is in practice mainly due to the paucity of information available. Since one of the compounding factors for the development of resistance is the concentration of a biocide, one can speculate that where a low concentration of a biocide is present, the resulting selective pressure will result in a change of (i) bacterial community, (ii) bacterial population or (iii) bacterial phenotype. However, without further evidence notably from in situ investigation, the overall risk of emerging resistance can only be assessed from in vitro derived evidence. It is also clear that a number of mechanisms will provide the bacteria with the ability to survive biocide and antibiotic exposure. If this has been demonstrated in laboratory investigations to some extent, there is an overall lack of information from the practice. However, when clinical and environmental isolates are investigated in laboratory investigations, these tend to show better survival ability to antimicrobials than their standard culture collection bacterial counterparts
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Sections 3.8.6.Tonnages and exposure &3.8. 7. Appearance of resistance in practice, p. 52-53
7.3 How can dissemination of resistance genes lead to resistance?
The SCENIHR opinion states:
3.9. Examples of biological hazards
The following sections present two possible events occurring amongst many. One is based on genetic dissemination of resistance genes, the other on the modification of the physiological state of the cells (biofilm).
3.9.1. Genetic dissemination of resistance genes
Mobile genetic elements (MGEs) play an important role in the evolution of bacteria. They allow the rearrangement or exchange of DNA between species, thereby increasing genetic diversity and flexibility of genomes (Dobrindt et al. 2004, Ochman et al. 2000). Among the various types of MGEs, genomic islands (GEI) take up a distinct position, because they are integrated in the chromosome of the bacterial host and thus potentially stably maintained. Those GEI that are mobile can excise from their chromosomal location, can induce self-transfer and reintegrate into a new host cell's chromosome are designated as integrated and conjugative elements. GEI can carry large regions (50–400 kb) with variable auxilliary functions that potentially benefit the host, such as growth in the presence of antibiotics or heavy metals, invasion of eukaryotic tissues via virulence factors, and exclusive growth with aromatic compounds (Dobrindt et al. 2004, Gaillard et al. 2008).
In a 2002 study, several staphylococcal clinical isolates resistant to the quaternary ammonium compound (qac)-based disinfectant benzalkonium chloride (83% of resistant strains exhibit plasmid-borne qacA/B and qacC genes), have been checked for antibiotic susceptibilities (Sidhu et al. 2002). A genetic linkage was reported between resistance to benzalkonium chloride products and penicillin and 44% of the plasmid-encoded ß-lactamase resistance was linked to disinfectant resistance genes. In addition, the frequencies of resistance to a range of antibiotics were significantly higher among qac-resistant than among qac-susceptible bacteria. Moreover, some isolates harbored multiresistance plasmids that contain qac, bla and tet resistant genes. The results are compatible with selective advantages of isolates carrying both disinfectant and antibiotic resistance genes and the data indicate that the presence of qac genes in staphylococci results in the selection of antibiotic-resistant bacteria (Paulsen 1998). Previous investigators have also reported a genetic linkage between disinfectant (qac) and antibiotic resistance genes (blaZ, aacA-aphD, dfrA, and ble) on the same staphylococcal plasmids from clinics and food environments (Sidhu et al. 2001, Sidhu et al. 2002) as well as the geographical dissemination of resistance genes among staphylococci (Bjorland et al. 2001, Noguchi et al. 2005). These conclusions are important because there are few investigations in this field.
The Salmonella genomic island 1 (SGI1) is an integrative mobilizable element originally identified in epidemic multidrug-resistant Salmonella enterica serovar Typhimurium DT104 (Doublet et al. 2003, Doublet et al. 2008). SGI1 contains a complex integron, which confers various multidrug resistance phenotypes due to its genetic plasticity. A multiple-antibiotic-resistant Salmonella enterica strain isolated from the environment was found to contain SGI1-K, a variant form of the Salmonella genomic island 1 (SGI1with an adjacent resistance module confering resistance towards mercury (Levings et al. 2007).
OqxAB, a plasmid-encoded multi-drug efflux pump identified in Escherichia coli of porcine origin and tested for substrate specificity, demonstrated a wide substrate specificity including animal growth promoters, antimicrobials, disinfectants and detergents (Hansen et al. 2005). The OqxAB pump can be transferred between Enterobacteriaceae (Salmonella Typhimurium, Klebsiella pneumoniae, Kluyvera sp. and Enterobacter aerogenes), conferring reduced susceptibility to various substrates including chloramphenicol, ciprofloxacin and olaquindox (Hansen et al. 2007).
Similar mobile elements containing biocide and antibiotic resistance genes have been reported in clinical isolates of another major human pathogen, Pseudomonas aeruginosa (Laraki 1999, Sekiguchi 2005, Sekiguchi 2007, Wang et al. 2007).
Consequently, the segregation/transfer of biocide and antibiotic resistance genes as integrative mobile genetic elements (MGEs) is a significant hazard for the selection and dissemination of MDR bacteria.
The uncontrolled use of biocides may recruit bacteria containing this type of genetic element and favor the vertical and horizontal spreading of the mobile elements to other bacteria (intra- or inter-specie) sharing the same ecological niches.
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Section 3.9.1. Genetic dissemination of resistance genes, p. 53 - 54
7.4 How can the formation of biofilms lead to resistance?
The SCENIHR opinion states:
Bacteria are able to adapt to shifts in nutrient availability, environmental stresses, and presence of inhibitory compounds as well as to immune defenses. One particularly important example of bacterial adaptation through systematised gene expression is the ability to grow as part of a sessile community, referred to as a biofilm. Biofilms are communal structures of microorganisms encased in an exopolymeric coat that form on both natural and abiotic surfaces (Hall-Stoodley et al. 2004). It is now recognized that biofilm formation is an important aspect of many, if not most bacterial diseases, including native valve endocarditis, osteomyelitis, dental caries, middle ear infections, medical device-related infections, ocular implant infections, and chronic lung infections in cystic fibrosis patients (Lynch et al. 2008)
When bacterial cells are in biofilm state, they demonstrate adaptive resistance in response to antimicrobial stress more effectively than corresponding planktonic populations. Antibiotic concentrations necessary to inhibit bacterial strains in steady-state biofilms were up to 10–1000 times greater than the concentrations needed to inhibit the same strains grown planktonically (Lewis 2001). Thus, in the presence of therapeutically available antibiotic concentrations, significantly higher proportions of the biofilms remained viable as the biofilms reached steady-state growth (Sedlacek and Walker 2007).
Moreover, bacteria inside biofilms resist better to biocidal agents. Examples are the reduced susceptibility to triclosan observed in Salmonella (Tabak et al. 2007) and in Proteus/Providencia (Stickler and Jones 2008, Williams and Stickler 2008), increased survival after exposure to quaternary ammonium compounds in Enterobacter sakasakii (Kim et al. 2007) and resistance to peroxides of Listeria cells in biofilms (Pan et al. 2006).
The resistance to clinically relevant antibiotics and to biocides could be related to common mechanisms which include: a localised high concentration of bacteria in the biofilm, modified physiological state of bacterial cell in the biofilm, decreased growth rate, restricted penetration of antimicrobials into a biofilm due to the presence of extracellular products (exopolymers and extracellular enzymes, and expression of possible resistance genes (Lewis 2001).
Although several authors report interaction between bacterial biofilm physiological state and resistance to antibiotics or biocide, and these resistances probably share common mechanisms, very little information is available on the cross resistance of sessile bacteria to antibiotics and biocide.
In one study (Jurgens et al. 2008), the aim of which was to determine if exposure of Pseudomonas aeruginosa biofilms to chloraminated drinking water could lead to individual bacteria with resistance to antibiotics, it has been demonstrated that exposure to chloramine does not increase antibiotic resistance in this bacterial species
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Section 3.9.2. Biofilms, p.54 - 55