Throughout the evolution of our species, life expectancy has generally been between 25 and 35 years with infection diseases being the balance needle. The living conditions of human beings progressively improved to the point that nowadays, a person born in a high-income country can expect to live more than 85 years (1). This result has been achieved primarily by improved hygiene, antibiotics and vaccination introduction. These three pillars have represented the most effective medical interventions to reduce mortality and morbidity caused by infectious diseases. In fact, in the poor areas of our planet where hygiene and the use of antibiotics and vaccination are not well-established, life expectancy is still lower than 50 years (2). Vaccination has protected humans by training the immune system to recognize and establish a rapid and effective response against a pathogen. In the 20th century, vaccines were introduced to large populations with the immunization practice developed by Edward Jenner. Since then, vaccination has become a widespread medical treatment determining the control of 16 major diseases, namely, smallpox, diphtheria, tetanus, yellow fever, pertussis, Haemophilus influenzae type b disease, poliomyelitis, measles, mumps, rubella, typhoid, rabies, rotavirus, hepatitis B, pneumococcal, and meningococcal diseases. Noteworthy is the complete or partial eradication achieved for smallpox and poliomyelitis, respectively (3).

To counteract infectious diseases, the discovery of antimicrobial treatment was another significant milestone that has dramatically reduced mortality. The modern era of antimicrobial therapy initiated in the 19th century with the identification of anti-syphilitic and anti-trypanosomal molecules derived from organic compounds chemically synthetized (4). In 1928, the discovery by Alexander Fleming of a new class of non-toxic antimicrobial agents derived from environmental fungi gave rise to the “golden era” of antibiotic discovery (1945–1960) (5). Conversely to active vaccination, drugs are therapeutics with different modes of action targeting the bacterial functions such as cell wall integrity, nucleic acid synthesis and repair, or protein biosynthesis. Moreover, drugs can be naturally produced by microorganisms (including environmental fungi and saprophytic bacteria), generated by chemical modifications of the natural antimicrobial agents or fully synthetized (6).

In combination with the vaccination practice, the discovery of antibiotics and their successful use in medicine is considered one the most relevant findings from a global health perspective (Figure 1). Nevertheless, the effectiveness of antibiotics has weakened to the point that our lives can be severely threatened. In fact, the antimicrobial resistance (AMR) is one of the most daunting problems that is causing the spread of infectious diseases and the increase in the number of deaths caused by infections that were previously considered uncomplicated (7). For example, the bloodstream infections caused by bacteria resistant to one or several drugs (multidrug-resistant; MDR) such as Pseudomonas aeruginosa are characterized by a 50% of mortality compared with the 24% of the non-multidrug-resistant infections (8). In addition, medical procedures such as surgeries, immunosuppressive chemotherapy and organ transplantation are becoming more critical and, in some cases, even prohibitive considering the need of effective antibiotics against multidrug-resistant pathogens. Therefore, the consequences of such microbial evolution can be dramatic with infectious diseases that could severely reduce our lifespan to an extent similar to the pre-antibiotic era. Globally, AMR pathogens are causing 700,000 deaths/year, and 10 million deaths/year are expected by 2050, a number even, higher than the 8.2 million caused by cancer today (9) (Figure 2).

Antibiotic resistance is considered nowadays as one of the greatest threats to human health (10). Cases of antibiotic resistance are constantly reported, and the time needed for bacteria to become resistant to newly introduced antibiotics, is getting shorter. In fact, antimicrobial use exerts evolutionary pressure for the creation and transmission of resistant pathogens, thus reducing antimicrobial effectiveness and raising the incidence of severe disease (11). However, this is not a new phenomenon and is commonly observed as soon as the introduction of new classes of antibiotics occurs (12). In 1946, Alexander Fleming anticipated this global burden with the renowned sentence “There is probably no chemotherapeutic drug to which in suitable circumstances the bacteria cannot react by in some way acquiring “fastness” [resistance]” (13). In fact, penicillin became commercially available in 1943 and resistance was observed for Staphylococcus aureus by 1948. In this context, the discovery by Barbara McClintok that transposons play a major role in the genomic diversity and evolution paved the way for a deeper understanding of the genetic basis underlying the antimicrobial resistance dissemination. Transduction, conjugation, transformation and other mobile genetic materials (transposons and integrons) are all possible mechanisms for the transmission of genetic determinants involved in AMR (14, 15) and in the generation of “superbugs” (16). Worryingly, the transmission of multiple resistance genes is not only relatively common among organisms within the same genus but also among evolutionarily distant organisms.

A plethora of mechanisms has contributed to the emergence of antimicrobial resistant microorganisms and to their spread. The first classification is between natural and acquired resistance mechanisms. The natural resistance relies on the bacterial ability to block the antibiotic function as a result of inherent structural or functional characteristics. For example, vancomycin (glycopeptide antibiotic) which inhibits peptidoglycan crosslinking, is generally effective only against Gram-positive but not Gram-negative bacteria, due to its inability to cross the outer membrane to get access to the d-Ala-d-Ala peptides. By contrast, the acquired resistance is based on the gaining of new functions; bacteria that would be originally susceptible, became resistant to one or more antibiotics (17). The main mechanisms exploited by bacteria to tackle the action of antibiotics are based on (i) the inactivation of the drug by its hydrolysis or structural modification, (ii) the prevention of the access to the target by reducing membrane permeability or overexpression of efflux pumps, (iii) changes in the antibiotic targets by mutation or post-translational modifications (Figures 3A–C) (18). The mechanisms of antibiotics inactivation comprise both the production of enzymes that destroys the antibiotic itself (for example, β-lactamases) and the production of enzymes that modify its structure (for example, aminoglycoside-modifying enzymes). β-lactam antibiotics, such as penicillin and cephalosporins, are hydrolyzed by a wide variety of β-lactamases. Since the early discovery of these enzymes, bacteria resistant to all β-lactam antibiotics have emerged (19, 20). In this regard, the genes encoding for extended-spectrum β-lactamases (ESBLs with activity against oxyimino-cephalosporins) and carbapenemases (21), including the IMP (imipenemase), VIM (Verona integron encoded metallo β-lactamase), K. pneumoniae carbapenemase (KPC) and OXA (oxacillinase) have been identified not only on the chromosomes, but also on plasmids as in the case of Enterobacteriaceae (22, 23) and of P. aeruginosa (8). Among them, there is the New Delhi metallo-β- lactamase 1 (NDM-1), an enzyme that confers high level of resistance to multiple antibiotics, including penicillin, cephalosporins and carbapenems and that is capable of rapid worldwide dissemination (14). This is of high concern since there are few available treatment options beyond the carbapenems. The main producers of the NDM-1 enzyme are Enterobacteriaceae such as Klebsiella pneumoniae, Escherichia coli and Proteus mirabilis, but also Acinetobacter spp. and Pseudomonas aeruginosa (Figure 3A) (24). Lastly, aminoglycoside-modifying enzymes are able to modify the structure of the antibiotic molecule by the transfer of different chemical groups such as acyl, phosphate, nucleotidyl and ribitoyl groups. This modification prevents the antibiotic from binding to its target protein mainly due to the steric hindrance (Figure 3A). For example, in 2012 a novel genomic island encoding six aminoglycoside-modifying enzymes, including gentamycin, was identified in Campylobacter coli isolated from broiler chickens in China (25) and more recently in Group B streptococcus (26). Yet, the prevention of antibiotic access to a specific target can occur by reducing the permeability of the membrane and/or increasing efflux in order to reduce the intracellular concentration of a drug (27, 28). Differently from Gram-positive bacteria, Gram-negative bacteria are intrinsically less permeable to many antibiotics as their outer membrane forms a sort of permeability barrier. Generally, the permeability is reduced by the downregulation of the major porins, which results in a decrease in antibiotics entry into the bacterial cell. Porins are beta barrel proteins that cross the cellular membranes acting as channels for passive diffusion of certain molecules (29). The expression or the structure of a specific porin can be influenced by mutations that decrease membrane permeability, reducing the entry of antibiotics inside the cell. For instance, P. aeruginosa OprD-deficient strains are characterized by high level of resistance to carbapenems (30). Bacterial efflux pumps belonging to the resistance-nodulation-division (RND) family are not selective for a specific class of antibiotic and confer intrinsic resistance to multiple and structurally distinct classes of drugs, including the β-lactams, quinolones, and aminoglycosides (31). The high-level expression of efflux genes is often due to mutations in their regulatory network that generally occur within a local repressor, a global transcription factor, promoter regions or intergenic sites. For example, by mutations and gene duplication mechanisms the overexpression of MexAB-OprM multidrug efflux systems confers to some P. aeruginosa isolates the ability to became resistant to carbenicillin antibiotics. Moreover, Nicoloff et al. identified a large tandem duplication of the E. coli acrAB locus in a mutant isolated in the presence of tetracycline (32). Such a mutation was found to overexpress the AcrAB drug efflux pump, producing an MDR phenotype (33). In N. gonorrhoeae, a mutation in the consensus −10 sequence upstream of mtrC strengthens its promoter activity determining constitutive overexpression of the efflux pump (34), (Figure 3B).

The other mechanism responsible for drug resistance is represented by the modification of the target by genetic mutations or post-translational modifications. Mutations in target genes can enable bacteria to overcome the toxic effects of antibiotics. Examples are provided by mutations in bacterial DNA gyrases (e.g. gyrA) and topoisomerase (e.g. parC) for fluoroquinolones, the β subunit of RNA polymerase (rpoB) for rifampin, 16S rRNA (rrs) for tetracyclines and aminoglycosides and 23S rRNA (rrl) for linezolid (13). The clinical use of linezolid has selected S. pneumoniae (35) and S. aureus (36) resistant lineages by a mutation in one of the rrl copies. The high recombination frequency has subsequently enabled the allele enrichment in the bacterial population. Another example of a target change is the acquisition of specific genes by horizontal gene transfer (HGT), such as in methicillin-resistant S. aureus (MRSA). In this case, the methicillin resistance is obtained by the acquisition of the staphylococcal cassette chromosome mec (SCCmec) element. This cassette encodes for a mutated form of PBP2 that is not recognized as a target by the β-lactamase enzyme but is still able to mediate a regular cell wall biosynthesis. Additional antibiotic resistance mechanisms consist in the protection by modification of the target molecule. This is exemplified by the polymyxin resistance in Acinetobacter baumanni (polymyxin B and polymyxin E, also known as colistin). The polymyxin antibiotics, are cyclic antimicrobial peptides with long, hydrophobic tails that bind to the lipopolysaccharide (LPS) of Gram-negative bacteria disrupting cell membranes. This resistance is often associated with changes in the expression of regulators affecting LPS production. For example, the addition of phosphoethanolamine (PE) to lipid A due to overexpression of pmrC results in a substantial reduction of colistin binding as the negative charge of the LPS is hugely lowered (37).

Given the alarming increase in antibiotic resistance cases, several human pathogens have been classified as a priority for the design of appropriate preventive and therapeutic strategies. In 2017, WHO published a prioritization list of pathogens (classified as critical, high and medium priority) to guide discovery, research and development of new antibiotic (38). In 2019 an estimation performed by the Centers for Disease Control and Prevention (CDC) in the United States reported that more than 2.8 million antibiotic-resistant infections occur in the U.S. each year, and more than 35,000 people die as a result of these infections. The antibiotic-resistant strains were classified into four categories according to the urgency of intervention (urgent, serious, concerning and watch list) (39). These bacteria can cause severe and often deadly infections and have become resistant to a large number of antibiotics, including carbapenems and third generation cephalosporins (the best available antibiotics for treating multidrug resistant bacteria). A relevant example is provided by A. baumannii multi-drug resistant isolates, that frequently cause outbreaks in hospitals. These strains are characterized by an extraordinary ability to rapidly alter their genome surviving on inanimate surfaces and medical equipments even in the presence of disinfectants (40). In A. baumannii, “resistance islands” located on the bacterial chromosomes and/or on mobile genetic elements induce a drug-resistant phenotype. For example, AbaR1 is an 86kb resistance- island generated by recurrent insertions of different mobile genetic elements, which can spread by horizontal gene transfer (HGT) within the genera Pseudomonas, Salmonella, and Escherichia. Genomic islands like AbaR1 confer resistance to various antibiotics by spreading the cluster of genes encoding for OXA-type beta-lactamases, aminoglycoside-modifying enzymes, proteins responsible for the modification of the lipid A and overexpression of the AdeABC efflux system (41). Tuberculosis (TB), even if not initially included in the WHO list, was already considered one of the most urgent priority because of its growing resistance to traditional treatment. TB now kills more people than any other pathogen (1.8 million in 2015) (42). Unfortunately, the resistance to isoniazid (INH), one of the four antibiotics used for the treatment of TB, is constantly increasing. INH leads to the inhibition of mycolic acids (MA) synthesis, the major lipid component of the mycobacterial cell envelope (43). The mechanisms of action of isoniazid are diverse and not fully known due to the involvement of a number of different genes. However, several studies suggest that the catalase KatG activates INH enabling the binding to NAD and the inhibition of enoyl acyl carrier protein (ACP) reductase (InhA). As a result, MA synthesis is blocked leading to mycobacteria death. When mutations in the katG gene occur, INH remains in an inactive form, unable to bind NAD and to inhibit InhA activity (Figure 3D). Consequently, InhA accomplishes its function in cell wall synthesis determining the survival of TB. This type of resistance is of concern as it negatively affects the outcome of TB-treatment facilitating the dissemination of MDR-TB and reducing the efficacy of INH (44). The second and third tiers in the list—the high and medium priority categories—include other increasingly drug resistant bacteria that cause more common diseases, such as Neisseria gonorrhoeae (the causative agent of gonorrhea) (45, 46). Since the treatment of N. gonorrhoeae with sulfonamides, this bacterium has acquired genetic resistance determinants capable to inhibit the killing by all major classes of antibiotics that are used as first line methods of treatment against gonorrhea infection (47). The main lethal target of cephalosporins is the transpeptidase penicillin binding protein 2 (PBP2), encoded by penA. Most of the resistant isolates contain mosaic mutations in penA that confer resistance to cephalosporins. In addition, the overexpression of efflux pump, MtrCDE, contributes to resistance through an increase of the drug efflux. The simultaneous mutations in the major porins PorB and mtrR, the repressors of MtrCDE efflux pump, represent an important resistance determinant of gonococcus. Hence, the outstanding capacity of N. gonorrhoeae to rapidly acquire resistance has led the World Health Organization and Center for Disease Control (US) to classify this bacterium as “superbug.” As a result, if new prophylactic and/or therapeutic strategies will not be discovered soon, we may enter an era of incurable gonorrhea infections (48). Therefore, understanding the mechanisms underlying the AMR will help to rationally design new preventive strategies to effectively tackle these dangerous threats for the human health.

Antibiotic resistance occurs when bacteria are not controlled nor killed by previously effective drugs through several mechanisms described above. Resistance is largely driven by the abuse of antibiotics in humans and animals, often with little or no therapeutic benefit. The use of antibiotics in viral respiratory infections or abuse in animal care and in agriculture to promote livestock growth, are common examples of the misuse (49). Additionally, the inability to rapidly identify the specific infecting pathogen at the point of care make the empiric treatment the only possible choice. In fact, the empiric treatment requires the use of broader spectrum antibiotics to cover all the key pathogens responsible for a particular infection (50). Moreover, the relationships between the human microbiome and the host immune system is hugely threatened. Antimicrobials alter the structure of the microbiota, expand the pool of antimicrobial-resistance genes, degrade the protective effects of the microbiota against invasion by pathogens, and may impair the efficacy of alternative medical treatments (51). Hence, the abuse of antibiotics has negatively affected the immune response making individuals more susceptible to infections. In fact, the pressure imposed by the antibiotic treatments on microbes other than the targeted pathogen (“bystander selection”) is hypothesized to be a major factor in the propagation of antibiotic resistance (11). International traveling is also a key factor in the dissemination of highly resistant strains. New Delhi metallo-β- lactamase 1 (NDM-1) and mcr-1 are the mobile resistance genes increasingly spreading worldwide and generating resistance to a variety of antibiotics including penicillins, cephalosporins and carbapenems. This is particularly relevant for infections caused by Gram-negative bacteria sensitive only to carbapenems, and for which only very few alternative possibilities are available. Furthermore, the NDM-1 resistance genes are transmitted very easily, as shown by the two Dutch travelers which were infected by a NDM-1-producing bacterial strain during their trip to India, even if not visiting any health centers (52). Enterobacteriaceae (Klebsiella pneumoniae, E. coli and Proteus mirabilis), Acinetobacter spp. and Pseudomonas aeruginosa are the most common NDM-1-producing bacteria for which colistin is considered the only possible therapeutic option. Nevertheless, with the spread of the mcr resistance gene (mcr-1) a generation of pan-resistant strains is arising. This has been probably due to the large use of colistin to increase pig growth in farms in China. In addition, to the raw meat, mcr-1 has been isolated also in samples from infected patients (53). The mcr-1 resistance determinant is able to easily spread in different bacterial species and also worldwide due to business globalization (24). Hence, the spread of resistant organisms, highly infectious and inducing high rate of morbidity and mortality has a strong economic impact, in terms of costs of treatment and long-term hospital stay, with a great burden on health systems.

The rising of AMR is a threat to modern medicine and new measures to control antibiotic-resistant bacteria are desperately needed including the development of new antibiotics. Nevertheless, the discovery of new effective chemical compounds with an appropriate balance of antibacterial activity, drug metabolism, pharmacokinetics properties and safety it is a daunting task. In fact, the pipeline run dry ~40 years ago (54). In addition, challenging logistics and high costs of large clinical trials make them nearly impossible to bring to market. Even if successful, the clinical utility of antibiotics will decline as resistance to them inevitably rises (55). Improving hygiene and the correct use of antibiotics while expanding their access in low- and middle-income countries are important tools to limit the burden of AMR, despite the fact these measures alone are not enough (50). Hence, vaccines may become a valuable and effective weapon to fight AMR. An important aspect is that resistance mechanisms are of less concern in vaccination. As reported above the mechanisms of resistance to antibiotics are mediated by the generation of spontaneous mutations or the acquisition of mobile genetic element by HGT. These mechanisms confer to bacteria the ability to tackle the killing effect of a drug and survive. Antibiotics are therapeutic measures since generally prescribed after the settling down of an infection when hundreds of millions of bacteria are infecting the body. On the contrary, vaccines are designed to prevent diseases. Their prophylactic use allows the host to build an immune response before encountering the pathogen or even at the beginning of an infection when only a few hundred or thousands bacteria are present. As the occurrence of resistance mechanisms can stochastically arise among billions of bacteria, it is evident that this is less likely to occur following vaccination. Furthermore, most antibiotics have a single target while vaccines have multiple targets inducing host-specific antibody and/or T cell responses. Even in this case, more mutations are likely needed to confer resistance to vaccines making the development of microbial resistance even harder (56, 57). Therefore, vaccines can be effective against antimicrobial resistance in different ways:

Rising antimicrobial resistance (AMR) is a threat to modern medicine and it also represents a major economic burden on healthcare systems. In the European Union, antibiotic resistant infections determine an economic burden of ~€1.5 billion per year. The US health system spends 20 billion USD in excess each year to treat resistant infections (102). By 2050, it is foreseen that USD100 trillion will be the economic damage due to AMR. According to the World Bank simulation, the global economy could lose as much as 3.8% of its annual gross domestic product by 2050 in a worst-case scenario. However, estimating the incidence and attributable mortality is challenging. This cost depends on a wide variety of factors: which drug and pathogen are involved, the mechanism of antibiotic resistance, the prevalence of that pathogen, the types of infections, the level of transmissibility, the health burden of those infections and whether alternative treatments are available. AMR can result in treatment failures and previously uncomplicated infections become more complex and severe (103, 104). It has recently been estimated that a 30% reduction in the efficacy of antibiotic prophylaxis for surgical procedures and chemotherapy would result in 120,000 additional infections and 6,300 infection-related deaths per year only in the United States. There is a risk of the cost being far higher than current best estimates. As already mentioned, vaccines represent a valuable and extraordinary tool to tackle the AMR problem. Besides being able to protect against serious diseases, vaccines may also reduce avoid mild disease episodes that may not receive medical attention, but which have important societal consequences. Vaccination decreases the disease incidence, the number of deaths, the longer hospital stays, and the need for more antibiotics if others fail owing to resistance. Unvaccinated individuals also benefit from wider societal benefits such as the lower transmission rates. This can be translated into a reduced risk of epidemics. In health economics, Incremental Cost–Utility Analysis (ICUA) is a method of financial analysis that can drive procurement decisions. The purpose of ICUA is to perform a cost-effectiveness analysis based on a careful evaluation of the ratio between the cost of an intervention and the benefit it generates in terms of increase in life expectancy. Value of vaccines and drugs is calculated based on the ICUA financial method resulting in an underestimation of the former. The added value of vaccines such as the induction of herd protection, reduction in time of hospitalization and reduction in anti-microbial resistance are difficult to measure in the short term. Therefore, since in the ICUA analysis, the benefits of medical interventions are evaluated during the period of medical observation, important benefits of vaccines which are only measurable in the long term are not considered. Although the ICUA analysis has generated more comprehensive models for financial evaluation, all the benefits of vaccination still need to be included in the analysis for the measurement of a full vaccine value (105). In 2015, the burden of resistant infections in countries of the European Economic Area (EEA) was measured in terms of number of cases, attributable deaths, and disability-adjusted life-years (DALYs) providing useful information for public health decision-makers prioritizing interventions for infectious diseases (106). The contribution of various antibiotic-resistant bacteria to the overall burden varies greatly between countries, thus highlighting the need for prevention and control strategies that are tailored to the needs of each country. Evaluation of the economic cost of AMR is important for decision-making and should be estimated accurately. Only combined efforts in the fields of antibiotics, new technologies for vaccine development and monoclonal antibodies will provide affordable solutions to tackle antimicrobial resistance.

SN, RRo, MP, and RRa wrote the manuscript and contributed to the ideas and concepts it contains. All authors reviewed and approved the manuscript.

RRo, MP, and RRa are permanent employees of the GSK group of companies. RRo, MP, and RRa are listed as inventors on patents owned by the GSK group of companies. SN is a PhD Research Fellow of University of Bologna (IT). This work was sponsored by GlaxoSmithKline Biologicals SA.