Introduction:
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:
Introduction:
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 that 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).
Plasmids, bacteriophages, and extracellular DNA are the three primary drivers of HGT through the processes of conjugation, transduction, and natural transformation, respectively. See Cameron
Conjugation through Plasmids: is considered the main recognized mechanism responsible for genetic material transfer in bacteria and for the emergence of multi-drug resistance in hospital environments and aquaculture, amongst others. Conjugation is one of the most active ways of gene transfer, and it is responsible for the propagation of different antibiotic resistance genes in the Enterobacteriaceae family, the conjugative plasmids being the most studied mobile genetic elements.
Bacterial conjugation, also referred to as bacterial sex, is a major horizontal gene transfer mechanism through which DNA is transferred from a donor to a recipient bacterium by direct contact. Conjugation is universally conserved among bacteria and occurs in a wide range of environments (soil, plant surfaces, water, sewage, biofilms, and host-associated bacterial communities). Within these habitats, conjugation drives the rapid evolution and adaptation of bacterial strains by mediating the propagation of various metabolic properties, including symbiotic lifestyle, virulence, biofilm formation, resistance to heavy metals, and, most importantly, resistance to antibiotics.
Conjugation was first discovered in 1946 by Edward Tatum and Joshua Lederberg, who showed that bacteria could exchange genetic information through the unidirectional transfer of DNA, mediated by a so-called F (Fertility) factor. It was later realized that the F factor is a replicative extra-chromosomal genetic element, for which they later coined the term plasmid, that can be transferred across the cell membranes of the parental strains. Since this seminal discovery, the identification of a plethora of conjugative elements, including plasmids, conjugative transposons, and integrative conjugative elements (ICEs), has revealed that conjugation is a universally conserved DNA transfer mechanism among Gram-negative and Gram-positive bacteria. See Lesterlin
Conjugative plasmids generally carry all the genes required for their maintenance during the vertical transfer from the mother to the daughter cells, as well as the genes necessary for horizontal transfer during conjugation from the donor to the recipient cell. These functions are encoded by different regions or modules that compose what is generally referred to as the plasmid backbone. Isolation and sequence analysis of an increasing number of conjugative plasmids has revealed considerable diversity in terms of genetic properties and organization. This diversity also indicates that different plasmids might use various regulations, molecular reactions, and strategies to achieve productive conjugational transfer and maintenance.
In contrast to transformation and transduction, conjugation requires direct cell–cell contact, with the interaction of a highly specialized structure called pilus, which is formed by a protein encoded in the plasmid termed pro-pilin that is part of the tubular structure of the pilus. This cell–cell interaction results in the unidirectional transfer of genetic material (from the donor cell to the recipient cell). This transfer is carried out with the formation of a bridge or conjugative pore that allows the communication between cellular cytoplasm of both cells. Once the recipient cell has acquired the new genetic material, it also acquires the genetic and phenotypic characteristics (encoded in the plasmid) including the transfer characteristics (tra genes).
The typical conjugative apparatus consists of four components: an origin of transfer (oriT), a relaxase, a type IV coupling protein, and a type IV secretion system (a tube-like structure called pilus that allows donors to contact recipients). Only a very small fraction of cells
carrying conjugative plasmids express the conjugation machinery, but if the plasmids provide a fitness advantage, the transconjugants can rapidly expand in the population. Between 35% and over 80% of E. coli isolates collected from different habitats have conjugative plasmids with genes that confer resistance to at least one antibiotic. Mating experiments conducted in laboratory culture have shown that the conjugation frequency is affected by multiple factors, including the nature of the recipient cells, growth phase, cell density, donor-to-recipient ratio, whether conjugation is conducted in liquid or solid media, carbon, oxygen, bile salts, metal concentrations, presence of mammalian cells, temperature, pH, and mating time. See Cortez-Cortez
—Plasmids are self-replicating mobile genetic elements. They are separate from the chromosome and contain a specific subset of genes from the bacterial genetic pool. Many plasmids conjugate between different bacteria, especially related ones, leading to intra- and inter-specific dissemination of plasmid-specific genes, for instance, antibiotic resistance genes. As a result, virtually identical plasmids are isolated repetitively in different bacterial species.
Plasmids are extrachromosomal genetic elements that replicate independently of chromosomes. Persistence of these selfish genetic elements is improved when they carry genes that are useful to the host cell, such as ARGs in the presence of antibiotics. Consequently, many different ARGs circulate on plasmids.
Plasmids disseminate through bacterial populations primarily through the process of conjugation. Conjugation requires physical contact between two cells in the same environment, followed by the formation of a bridge that enables the transfer of a plasmid from a donor to a recipient cell
Bacterial transformation is the genetic alteration in a cell as a result of the direct absorption,
incorporation, and expression of exogenous DNA between closely related bacteria, and it is mediated chromosomally by encoded proteins. This foreign genetic material is ‘naked’ and can be present in the environment in which the bacterium thrives, and it can penetrate the bacterial cell membrane when the bacterium is in a ‘competent’ state, either due to lack of nutrients or elevated cell density. In order for the transformation to happen, the DNA must be transferred from the surface to the cytoplasmic membrane and then cross the cytoplasmic membrane through a highly conserved membrane channel.
This natural transformation is distinct from artificial transformation techniques involving electroporation and heat-shock treatments with chemically modified (e.g. CaCl 2 and MgCl2) cells that are routinely used for molecular biology applications. Transformation competence can be naturally or artificially induced, but not all bacterial species develop natural competence. In naturally transformable bacteria, competence is usually transient and induced by alterations in the growth state of organism. A group of “competence genes” has been identified, and general mechanistic models have been proposed. In general, Escherichia coli is not believed to be naturally transformable; it develops high genetic competence only under artificial conditions, including exposure to high Ca2+ concentrations and temperature shock. However, reportedly, E. coli can express modest competence under certain conditions that are feasible in its natural environments.
In many naturally competent bacterial species type-IV pili (T4P) are required for natural transformation. While the exact role of T4P in natural transformation is unclear, the generally accepted model is that DNA binds to the pilus structure, which retracts and pulls the DNA to the cell surface. It is unclear whether or not DNA is translocated across the outer membrane through the PilQ secretin pore.
In Gram-positive bacterial species that do not produce T4P, natural transformation involves a number of proteins with homology to T4P proteins, which are thought to form a pseudopilus
structure that spans the cell wall and is coupled to the DNA translocation complex at the cytoplasmic membrane.
Once exogenous DNA has been taken up by the cell it can be stably incorporated into the genome via recombination or transposition, or be maintained as an independent replicon if
plasmid DNA is taken up by an appropriate host.
Transduction through bacteriophages: Another HGT mechanism is transduction, in which DNA transfer is mediated by independently replicating bacteriophages, bacterial viruses that can package segments of host DNA in their capsid, and inject it into a new host when an environmental stimulus triggers cell lysis. When this happens, the new injected genetic material in the cell infected by the virus can be recombined with the chromosomal DNA, generating either a lytic or lysogenic cycle.
Bacteriophages (or phages) are viruses that infect bacteria. The phage–host relationship is specific and complex. Receptor-binding proteins (RBPs) of phages such as tail fibers and tail spikes are the first phage proteins interacting with the host, initiating the infection process. These proteins can specifically bind outer cell wall structures of bacteria such as capsular polysaccharides (CPS), or lipopolysaccharides (LPS), (lipo)teichoic acids, outer membrane proteins, flagella, or pili. Whereas most phages encode a single or two RBPs, some polyvalent phages express multiple RBPs, forming a branched RBP structure. Each of these RBPs
recognizes a different receptor, allowing the phage to infect multiple hosts.
Transduction occurs when viral particles transfer bacterial genes. After infection with a bacteriophage, bacterial DNA is sometimes accidentally packaged in a bacteriophage capsid. A capsid containing bacterial DNA is fully capable of binding to a recipient cell and injecting the foreign DNA. If the transferred bacterial DNA is recombined into the genome of the recipient cell, transduction has occurred.
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.
Vancomycin Resistance: Emergence of vancomycin resistance in enterococci was reported in 1986, approximately 30 years after the introduction of this antibiotic into clinical practice. More recently, vancomycin resistance was detected in strains of Staphylococcus aureus, Oerskovia turbata, Arcanobacterium haemolyticum, Streptococcus bovis, Streptococcus gallolyticus, Streptococcus lutetiensis, Bacillus circulans, Paenibacillus, and Rhodococcus, as
well as in anaerobic bacteria belonging to the Clostridium genus and Eggerthella lenta.
A primary mechanism of vancomycin resistance is the modification of the antibiotic’s target molecule in bacteria, which reduces the drug’s ability to bind and function. This typically involves replacing the target dipeptide precursor, Dcap D𝐷-Ala-Dcap D𝐷-Ala, with Dcap D𝐷-Ala-Dcap D𝐷-Lac or cap D𝐷-Ala-Dcap D𝐷-Ser, which has a much lower binding affinity for vancomycin. Other mechanisms can also contribute, such as thickening the bacterial cell wall or creating “false binding sites” that trap the antibiotic.
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)).
P. aeruginosa for example has multiple enzymes that are common in most isolates of P. aeruginosa. AmpC Beta-Lactamases are chromosomally encoded enzymes that give resistance to penicillins, second- and third-generation cephalosporins, and cephamycins. See Schwartz
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)