Resistance Mechanisms

A large variety of antimicrobial resistance mechanisms have been identified, which can be classified into four major categories (Figure 3.1): (1) the antimicrobial agent can be prevented from reaching its target by reducing its penetration into the bacterial cell; (2) the antimicrobial agent can be expelled out of the cell by general or specific efflux pumps; (3) the antimicrobial agent can be inactivated by modification or degradation, either before or after penetrating the cell; and (4) the antimicrobial target can be modified or protected by another molecule preventing access of the antimicrobial to its target, so that the antimicrobial can no longer act on it. Alternatively, the antimicrobial agent target can be rendered dispensable by the acquisition or activation of an alternate pathway by the microorganism. A few examples of each of these resistance mechanisms are listed in Table 3.1 and more systematic information can be found in the following chapters of this book.

Types of Antimicrobial Resistance

In the context of antimicrobial resistance, bacteria display three fundamental phenotypes: susceptibility, intrinsic resistance, or acquired resistance.

Intrinsic resistance is natural to all the members of a specific bacterial taxonomic group, such as a bacterial genus, species, or subspecies. Guidance documents on known intrinsic resistance phenotypes are provided by the European Committee on Antimicrobial Susceptibility Testing¹ (EUCAST). This type of resistance occurs most often through structural or biochemical characteristics inherent to the native microorganism. For example, many Gram‐negative bacteria are naturally resistant to the activity of macrolides since these chemicals are too large to traverse the cell wall to gain access to their cytoplasmic target. Other examples include the general reduced activity of aminoglycosides against anaerobes, because of the lack of aminoglycoside uptake into the cells under anaerobic conditions, and polymyxin resistance among Gram‐positive bacteria because of the lack of phosphatidylethanolamine in their cytoplasmic membrane. These intrinsic resistances should generally be known by clinicians and other users of antimicrobial agents so as to avoid inappropriate and ineffective therapeutic treatments (Chapter 2).

Antimicrobial resistance is defined as acquired when a normally susceptible organism develops resistance through different types of genetic modification. Acquisition of resistance usually leads to discrete jumps in the MIC of an organism and hence to clear bi‐ or polymodal distributions of MICs.² However, in some instances such as for fluoroquinolones, acquisition of resistance (elevated MICs) may be a progressive phenomenon, through the step‐wise accumulation of multiple genetic modifications blurring the minimal changes in MIC provided by each modification into a smooth continuous MIC distribution curve (Bhatnagar and Wong, 2019).

Acquired resistance can be manifested as resistance to a single agent, to some but not all agents within a class of antimicrobial agents, to an entire class of antimicrobial agents, or even to agents of several different classes. In the great majority of cases, a single resistance determinant encodes resistance to one or several antimicrobial agents of a single class of antimicrobials (such as aminoglycosides, beta‐lactams, fluoroquinolones) or of a group of related classes of antimicrobials such as the macrolide‐lincosamide‐streptogramin B group. However, some determinants encode resistance to multiple classes. This is, for example, the case for determinants such as the Cfr rRNA methyltransferase (Long et al., 2006) or the aminoglycoside acetyltransferase variant Aac(6′)‐Ib‐cr (Robicsek et al., 2006), or when multidrug efflux systems such as the AcrAB‐TolC system in Enterobacterales are upregulated, resulting among others in reduced susceptibility to beta‐lactams, tetracyclines, chloramphenicol, and fluoroquinolones (Ebbensgaard et al., 2020). The simultaneous acquisition of several unrelated genetic resistance determinants located on the same mobile genetic element is, however, more common as an explanation of multidrug resistance.

Acquisition of genetic determinants of resistance is associated with a variety of MICs and does not always lead to clinically relevant resistance levels. A clear distinction should be made between epidemiological cut‐off values and clinical breakpoints, based on the presence of acquired mechanisms causing decreased susceptibility to an antimicrobial or clinical responsiveness, respectively (Chapter 2) (Bywater et al., 2006). In order to avoid many apparent contradictions and compromises between clinicians, microbiologists, and epidemiologists, the word “breakpoint” should only be used for clinical situations, while epidemiological cut‐off values should be used for the discrimination of wild‐type versus non‐wild‐type population.

Acquisition of Antimicrobial Resistance

Bacterial antimicrobial resistance can result from the mutation of genes involved in normal physiological processes and cellular structures, from the acquisition of foreign resistance genes, or from a combination of these mechanisms. Mutations occur continuously but at relatively low frequency in bacteria, thus leading to the occasional random emergence of resistant mutants. However, under conditions of stress (including those encountered by pathogens when facing host defenses or in the presence of antimicrobials), bacterial populations with increased mutation frequencies can appear (Couce and Blázquez, 2009). This so‐called mutator state has been suggested to be involved in the rapid development of resistance in vivo during treatment with certain antimicrobials such as fluoroquinolones. However, for the majority of clinical isolates, antimicrobial resistance results from acquisition of extrachromosomal resistance genes.

Foreign DNA can be acquired by bacteria in three different ways (Figure 3.2): (1) uptake of naked DNA present in the environment by naturally competent bacteria (called transformation); (2) transfer of DNA from one bacterium to another by bacteriophages (transduction); and (3) transfer of plasmids or related elements (such as integrative conjugative elements or ICEs) between bacteria through a mating‐like process called conjugation. The term mobilome describes all mobile genetic elements that can move around within or between genomes in a cell (Siefert, 2009). These have been divided into four classes: (1) plasmids and ICEs (Burrus and Waldor, 2004; Madec and Haenni, 2018); (2) transposons (Muñoz‐López and García‐Pérez, 2010); (3) bacteriophages (Chiang et al., 2019); and (4) self‐splicing molecular parasites (Edgell et al., 2011). Although there are some examples of bacteriophage‐mediated antimicrobial resistance transfer, the plethora of examples of transferable resistance plasmids found across a broad variety of bacterial hosts suggests that plasmids and conjugation are more important in the global spread of antimicrobial resistance genes in bacterial populations.

Plasmids are extrachromosomal self‐replicating genetic elements that are not essential to survival but that typically carry genes that impart some selective advantage(s) to their host bacterium, such as antimicrobial resistance genes. Despite the apparent efficiency of these transfer mechanisms, bacteria possess a large variety of strategies to avoid being subverted by foreign DNA, so that numerous obstacles have to be overcome to allow the stabilization and expression of genes in a new host (Thomas and Nielsen, 2005). In addition, plasmids compete for the replication and partition machinery within cells and plasmids that make use of similar systems cannot survive for long together in the same cell. This “incompatibility” has led to the classification of plasmids into so‐called incompatibility groups, a system based on the replicon type widely used to categorize resistance plasmids into similarity groups and to study their epidemiology (Carattoli et al., 2005). Another method of plasmid characterization, named “degenerate primer MOB typing” (DPMT), is based on the sequence of relaxase enzymes needed to nick one strand of the plasmid DNA and initiate its transfer. It allows the determination of MOB families that are partially overlapping with the incompatibility groups (Alvarado et al., 2012).

Many studies have shown that antimicrobial resistance plasmids can be transferred between bacteria under a wide variety of conditions. This includes, for example, the relatively higher temperature of the intestine of birds compared to other animals, as well as the lower temperatures encountered in the environment. Some plasmids can be transferred easily between a variety of bacterial species, for instance between harmless commensal and pathogenic bacteria, thus leading in some cases to the emergence and massive establishment of newly resistant pathogen populations in individual animals within days (Poppe et al., 2005).

In addition to moving between bacteria, resistance genes can also move within the genome of a single bacterial cell and hop from the chromosome to a plasmid or between different plasmids or back to the chromosome, thus allowing development of a variety of resistance gene combinations and clusters over time. Transposons and integrons play a major role in this mobility within a genome. Transposons (“jumping genes”) are genetic elements that can move from one location on the chromosome to another; the transposase genes required for such movement are located within the transposon itself. The simplest form of a transposon is an insertion sequence (IS) containing only those genes required for transposition. Composite transposons consist of a central region containing genes (passenger sequences) other than those required for transposition (e.g., antimicrobial resistance) flanked on both sides by ISs that are identical or very similar in sequence. A large number of resistance genes in many different bacterial species occur as part of composite transposons (Tansirichaiya et al., 2019).

Homologous recombination between similar transposons within a genome also plays an important role in clustering passenger sequences such as antimicrobial resistance genes together on a single mobile element. Some bacteria (mainly anaerobes and Gram‐positive bacteria) can also carry so‐called integrative and conjunctive elements (ICEs), which are usually integrated in the bacterial chromosome but can be excised, subsequently behaving like a transferable plasmid and finally reintegrating into the chromosome of their next host (Burrus et al., 2002).

The magnitude of resistance development is also explained by the widespread presence of integrons, particularly class 1 integrons (Ghaly et al., 2017). These DNA elements consist of two conserved segments flanking a central region in which antimicrobial resistance “gene cassettes” can be inserted. More than 130 distinct cassettes conferring resistance to numerous classes of antimicrobial drugs and quaternary ammonium compounds have been identified to date, and the list is likely to increase thanks to informatics tools allowing the detection of integrons from NGS data (Cury et al., 2016). One‐ended transposition through ISEcp1 or ISCR elements, which also helps mobilize adjacent genetic material by mechanisms different from classic insertion sequences, has been detected increasingly in relation with integrons and antimicrobial resistance genes (Toleman and Walsh, 2011). In addition, integrons are usually part of composite transposons, thus further increasing the mobility of resistance determinants.

The Origin of Resistance Genes and Their Movement across Bacterial Populations

Resistance genes and DNA transfer mechanisms existed long before the introduction of therapeutic antimicrobials into medicine, since antimicrobial‐resistant bacteria and resistance determinants have been found in Arctic ice beds estimated to be several thousand years old (D’Costa et al., 2011). Molecular characterization of the culturable microbiome of Lechuguilla Cave, New Mexico (from a region of the cave estimated to be over 4 million years old), revealed the presence of bacteria displaying resistance to a wide range of structurally different antimicrobials (Bhullar et al., 2012).

It is widely believed that antimicrobial resistance mechanisms arose within antibiotic‐producing microorganisms as a way of protecting themselves from the action of their own antibiotic, and some resistance genes are thought to have originated from these organisms. This has been substantiated by the finding of aminoglycoside‐modifying enzymes in aminoglycoside‐producing organisms that display marked homology to modifying enzymes found in aminoglycoside‐resistant bacteria. However, as in the case of synthetic antimicrobials such as trimethoprim and sulfonamides, preexisting genes with other resistance‐unrelated roles might have evolved through adaptive mutations and recombinations to function as resistance genes.

Some suggest that in their original host, antimicrobial resistance genes play a role in detoxification of components other than antimicrobials, and in a variety of unrelated metabolic functions (Martínez, 2008). A vast reservoir of such genes, now dubbed the resistome, is present in the microbiome of various natural environments, which can be transferred to medically relevant bacteria through genetic exchange (Wright, 2010). There is growing evidence that resistance genes identified in human bacterial pathogens were originally acquired from environmental, nonpathogenic bacteria via horizontal gene transfer (HGT), as has been exemplified for CTX‐M and mcr genes, which originated from Kluyvera spp. and Moraxella spp., respectively (Cantón et al., 2012; Kieffer et al., 2017).

It is now largely admitted that the epidemiology of antimicrobial resistance goes beyond the boundaries of veterinary and human medicine and must be tackled through a “One Health” approach (McEwen and Collignon, 2018). Despite these One Health approaches, the complexity of movement of microorganisms and of HGT involved inside and between all sectors is difficult to comprehend (Figure 3.3).

Because of the intricacies of the resistance issue, numerous strategies to control the rise of antimicrobial resistance at every level have emerged in the scientific and medical communities. As with other complex issues that global society faces, no single intervention will be decisive alone, but numerous interventions at both international and local scales are needed that cumulatively may preserve acceptable levels of efficacy for current and future antimicrobial drugs.

The Effects of Antimicrobial Use on the Spread and Persistence of Resistance

The increased prevalence and dissemination of resistance is an outcome of natural selection – the Darwinian principal of “survival of the fittest.” In any large population of bacteria, a few cells that possess traits that enable them to survive in the presence of a toxic substance will be present. Susceptible organisms (i.e., those lacking the advantageous trait) will be eliminated, leaving the remaining resistant populations behind. With long‐term antimicrobial use in a given environment, the microbial ecology will change dramatically, with less susceptible organisms becoming the predominant population (Andersson and Hughes, 2011). When this occurs, resistant commensal and opportunistic bacteria can quickly become established as dominant components of the normal flora of various host species, displacing susceptible populations. Changes in antimicrobial resistance frequency when new antimicrobials appear on the market or when restrictions are implemented in the use of existing antimicrobials support the validity of these evolutionary rules. Several examples of the rise and fall of antimicrobial resistance as selection pressures change are described later in this chapter.

The clustering of multiple resistance genes on plasmids, transposons, and integrons makes the problem of antimicrobial resistance even more challenging. When resistance genes are physically linked together or to other selectively advantageous genes, co‐selection will lead to the persistence of all the resistance genes as part of the cluster. Consequently, exposure to one antimicrobial will co‐select for bacteria that are also resistant to several unrelated agents. Examples of co‐selection are known, such as the maintenance of glycopeptide resistance in porcine enterococci by the use of macrolides (Aarestrup et al., 2000) or the emergence of gentamicin resistance due to the use of spectinomycin (Chalmers et al., 2017). Resistance to biocides can also trigger cross‐resistance to antimicrobials such as fluoroquinolones (Buffet‐Bataillon et al., 2016), while antimicrobial resistance can induce tolerance to biocides such as chlorhexidine (Liu et al., 2017; Wand et al., 2017). Likewise, heavy metals such as zinc can easily co‐select methicillin‐resistant Staphylococcus aureus (MRSA) isolates, since the czrC gene is inserted inside the SCCmec element (Cavaco et al., 2011

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