Antimicrobial resistance (AMR) and persistence are associated with an elevated risk of treatment failure and relapsing infections. Alhtough AMR has been widely acknowledged as a pressing issue, the incidence of infections and spread of MDR bacteria is still rising. In addition, the increased use of implanted medical devices such as joint prostheses, artifical heart valves, vascular endoprostheses and pacemakers leads to a higher incidence of biofilm assocaited infeciton, which in turn leads to anotehr important phenomenon; antibiotic tolerance. Antibotic resistance frequenlty resutls in dealyed adequate antibiotic treatment, increasing morbidity and mortality. AMR is the inherited ability of microorganisms to grow at high antibiotic concentration. It is usually quantified by measuring the mininum inhibitory concetnraiton (MIC) of a particular antibiotic wherein resistant bacteria are able to multiply and grow at concetnrations of antibiotics, which are fatal to other strains of the same species. In general, Gram-negative bacteria are less permeable than Gram-positive and show an intrinsic resistance to many antibacterial compounds such as the cell was acive glycoppetide vancomycin. These large molecules are unable to cross the outer bacterial membrane of Gram-negative bacteria and thus cannot target the cell wall of Gram-negative bacteria. In contrast, the Gram-positive bacteria Staphylococcus aureus is naturally susceptible to almost every antibiotic that has been developed, but it is well known for quickly acquiring antibiotic resistance by emans of botaining specific genetic modifications. (Huemer, “Antibiotic resistance and persistence – Implications for human health and treatment perspectives” EMBO Reports, 21, 2020).

Essentially any of the accessory genetic elements found in bacteria are capable of acquiring r genes and promoting their transmission; the type of element involved varies with the genus of the pathogen. In the streptococcie, miningococci and related genera, the exchange of both virulence and pathogenicity genes is highly promiscuous; the principal mechanisms for DNA traffic appears to be transformation. (Davies, “origins and Evolution of Antibiotic Resistance” Microbiology and Molecualr Biology Reviews, 2010, p. 417-433).

Antibiotic presistence or heterotolerance is defined as the ability of a bacterial subpopulation to surive high bactericidal drug concentrations to which the bacteria are fully susceptible. One major chacteristic of antibiotic peristence is a biphasic killing, with a rapid killing of the susceptible populations and a slower killing of the persister subpopulation. Persistent infections are ongoing infections, which are not cleared by the host, whereas antibiotic persistence is used to describe a bacterial population that suvives antibiotic exposure wihtout being resistant. (Huemer, “Antibiotic resistance and persistence – Implications for human health and treatment perspectives” EMBO Reports, 21, 2020)

To combat damage to themselves by antibiotics, bacteria have evolved stratgies that include both intrinsic and acquired resistance. Intrinsic resistance is ahceived by invactivaiton of antibiotics by hydrolytic or chemically modified enzymes, modification of antibiotic targets, cell membrane permeability barriers and antibiotic efflux pumps. In contrast, acquired antibiotic resistance is acieved through HGT, including transformation (bacteria absorb naked DNA for the eternal environment and insert it inot the genome), transduciton (chromosomal and extrachromosomal DNA is transferred between donor and recipitn bacteria via a bacteriophage intermediate), conjugation (mobile genetic elements such as plasmids and integratie and conjugative elements (ICEs) are transferred form one bacterium to another) adn membrane fesicle transport (vesicle-mediated transfer of DNA). These stregies ahve greatly helped bacteria to acquire resistance to antibiotics adn certian extensively ARB such as carbapenemase-producing colistin-resistant Klebsiella pneumonaie are now resistant to almost all antibotics.

Horizontal Gene Transfer:

Acquisition of foreign DNA material through horizontal gene transfer (HGT) is one of the most important drivers of bacterial evolution and it is frequently responsible for the development of antimicrobial resistance. Most antimicrobial agents used in clinical practice are (or derive from) products naturally found in the environmment (mostly soil). Bacteria sharing the environment with these molecules harbor intrinsic genetic determinants of resistance and there is robust evidence suggesting that such environmental resitome is a prolific source for the acquisition of antibiotic resistance genes in clinically relevant bacteria. Classically, bacteria acquire external genetic material through three main strategies (i) transformation (incorporation of naked DNA), ii) transduction (phage mediated) and, (iii) conjugation (bacterial “sex”). (Arias, “Mechanisms of Antibiotic Resistance” Microbiol Spectr, 2016, 4(2)).

The ecological role of naturally produced antibiotics should be to inhibit competitors. Given this role, resistance genes should have evolved to counteract antimicrobial action, so taht the function of each of these determinants is to avoid the activity of one specific antibotic (or a family of antibiotics with similar structures). Work with genes acquired by horizonatal gene transfer (HGT) confirm this idea. Plasmid-encoded beta-lactamases serve to resist beta-lactam antibiotics and are not active agaisnt other kinds of drugs. The same applies for mutation-drived resistance; quinolone reistance is the consequence of mutations in topoisoerases genes, which do not alter bacterial susceptibility to other antibotics. The only impact that hte acquisition of resistance is usually supposed to have on bacterial physiology is to produce a general metabolic burden. (Rojo, “Metabolic regulation of antibiotic resistance” FEMS Microbiol Rev 35 (2011) 768-789)

Mutations/modifications of an antibiotic target molecule:

Spontaneous mutations or post translational modifications of an antibiotic target molecule can elad to conformational changes that result in ineffective target binding and attenuation of antibiotic activity.

–Mutations in ribosomal protein genes: 

Ability to pump antibiotics out of cells: The production of complex bacterial machineries capable to extruTetracycline resistance is one of thede a toxic compound out of the cell can result in antimicrobial resistance.  classic examples of efflux-mediated resistance, where the Tet efflux pumps extrdue tetracyclines using protein exhcange as the source of energy. Currenlty, more than 20 different tet genes have been described, most of which are harbored in MGEs. (Arias, “Mechanisms of Antibiotic Resistance” Microbiol Spectr, 2016, 4(2)).

Decreased permeability of the bacterial cell membrane: Intracellular concentrations of antibiotics can be kept low via decreased permeability of the bacterial cell membrane, such as by reducing or mutating proins that would allow entry of antibiotic into the cell. (Huemer, “Antibiotic resistance and persistence – Implications for human health and treatment perspectives” EMBO Reports, 21, 2020)

Modifying antibiotics: Antibiotics can be inactivated by the transfer of chemical groups to vulnerable sites thus inhibiting target binding and function or directly destroyed by hydrolysis. Inactivation of antibiotics by addition of a chemical group, such as acyl, nucleotidyl, phosphate or ribitoyl groups, resulting in steric hindrance can casue antibiotic resistance.

Bacteria can also become resistant to antibiotics by the production of enzymes that digest/metabolize the antibiotic. (Popl, “ARDB-Antibiotic Resistance Genes Database” Nucleic Acids Research 2009, 37)

The main mechanisms fo beta-lactam resistance relies on the destruction of these compound by the action of beta-lactamases. These enzmes destroy the amide bond of the beta-lactam ring, rendering the antimicrobial ineffective. Beta-lactamases were first described in the early 1940s one year before penicillin was introduced to the market; however, there is evidence of their existence for millions of years. (Arias, “Mechanisms of Antibiotic Resistance” Microbiol Spectr, 2016, 4(2)).

Phenotypic Resistance:

Phenotypic resistance refers to those transient situations in which a bacterial population, otherwise susceptible to a given antibiotic, is refractory to its action. This transient resistance does not require a genetic change and thus it is not inheritable. The growth rate is the first parameter that impacts the phenotype of susceptibility to antibiotics of bacterial populations. The relevance of growth rate on the activity of penicillin was already described in 1944, in a study that showed that the activity o this antibotic was impared when cells grew slowly. However, this effect is not restricted to just beta-lactam antibiotics. It has been shown that resting cells are fully resistant to ampicillin or to tetracylcine, whereas streptomycin or ciprofloxacin is still active agaisnt cells in the stationary phase, although their activity is lwoer than that obeserved for exponentially growing bacteria. This situation has been called “drug indifference” and can be relevant for the persistence of bacterial infections even in patients under antibiotic treatment, when bacterai are in a host location that restricts grwoth or when the microoriganisms have ocnsumed the host resources and their growth is impaired. This can be particulalry important in the case of long lasting infections, becasue it was demonstrated that the concentraiton of antibiotics required to cure an experimental infection increases with the duration of the infection. The existance of resting or slow-growing cells which are refractory to treatment, is supposed to be the reason for the need to use prolonged regimes for treating infections by organisms such as Mycobacterium tuberculosis where hypoxia triggers dormancy and TB the organisms undergoes significant metabolic reprograming, with upregulation of stress-related gens and down-regulation of many central metabolism pathways. (Rojo, “Metabolic regulation of antibiotic resistance” FEMS Microbiol Rev 35 (2011) 768-789)

Metabolic Shifts:

Two other situations that might confer phenotypic resistance are persistence and growth in biofilms. Whereas it was first propsoed that this situation might be due to the existence of a fraction of nongrowing cells into any bacterial population that is phenotypically resitant to antibiotics, more recent work indicates that there are several difference mechanisms that can lead to prersistence. Metabolic enzymes, global regulationrs and toxin-antitoxin systems are among those elements that are relevant for devleoping persiter pehnotype. Among genes coding for metabolic enzymes, it was ofund that the lnockout of ygfA, which codes for a putative t-formyl-THF cycloligase involved in folate biosynthesis or of yigB, which may clock metabolism by depleting the pool fo flavin monoculcoetide, dereases persistence. Folate deficiency impaired the biosynthesis of purines, thymidilate and methionine. Overepxression of YgfA increased tolerance to ofloxacin. These reuslts support the existence of a linkdage between some specific metabolic pathways and bacterail persistence. Detailed anlsyes of P. aeruginosa biofims showed that the response to antibiotics was highly dependent on the metabolic state of each population. For instance, aerobically growing bacteria were sensitive to ciprofloxacin, but resistant to polymyxin, whereas the opposite was found for cells grwoting in the deepest part of the biofilm, which present an anaerobic metabolism. While some metabolic shifts can make bacteria phenotypically resistant to a particular antibotic, the opposite might occur as well where some specific grwoing conditions can make bacterai more susceptible during infection than when growing in vitro. For example, Listeria monocytogenes is an intracellular pathogen that grows inside mammal cells using hexose phosphates present in the host cytosol. For frwoing into host cells, Listeria upregulates a set of virulence detemrinatns, whose expression is under control of the transcriptional regulator PrfA. One of the PrfA0regulated determiants required for intracellular growth is the sugar phosphate transporter Hpt. This transporter is highly expressed during Listeria intracellular growth, but its expression is very low when bacteria grwo in vitro in the media regulalry used for suspectiblity tests. (Rojo, “Metabolic regulation of antibiotic resistance” FEMS Microbiol Rev 35 (2011) 768-789)

Given that antibiotic susceptbility can change as a consequence of laterations in the bacterail metabolism, it is conceivable that bacterail colonization of novel habits might select antibiotic resistance even int he absence of antibiotic selective pressure. One of the best known chronic infectious diseases is cystic fibrosis, which is the most prevalent inherited disease in the Caucasion population. CF pateitns suffer chronic infections in their lungs by different bacterail species such as P. aeruginosa. (Rojo, “Metabolic regulation of antibiotic resistance” FEMS Microbiol Rev 35 (2011) 768-789)