The European Medicines Agency Approved the New Antibacterial Drugs – Response to the 2017 Who Report on the Global Problem of Multi-Drug Resistance

According to the World Health Organization (WHO), the growing drug resistance of microorganisms is one of the top ten threats to humanity. In 2017, the WHO published a special report containing a list of pathogens for which the search for new therapeutic options is a priority, due to the increasingly limited range of antibiotics that can be used to treat infections caused by them [121]. Twelve pathogens were entered on the list, divided into 3 categories according to the urgency of searching for new therapeutic options active against them. The first group of pathogens with the highest critical priority included carbapenem-resistant Gram-negative bacilli. Carbapenem-resistant Acinetobacter baumannii (CRAB) strains were considered to be the most dangerous, followed by carbapenem-resistant Pseudomonas aeruginosa strains CRPA) and, in third place, carbapenem-resistant Enterobacterales (CRE). This group also includes Enterobacterales strains resistant to third-generation cephalosporins, as well as Mycobacterium tuberculosis and other acid-fast mycobacteria. Whereas, multidrug-resistant strains of Gram-positive cocci have been classified by the WHO into the second group of pathogens with a high priority of search for new drugs against them. This group includes Staphylococcus aureus strains resistant to methicillin (methicillin-resistant S. aureus – MRSA) and vancomy-cin (vancomycin-resistant S. aureus – VRSA);vancomycin-resistant Enterococcus faecium, as well as clarithromycin-resistant Helicobacter pylori; Campylobacter spp., and Salmonella spp. resistant to fluoroquinolones; and Neisseria gonorrhoeae resistant to fluoroquinolones and third-generation cephalosporins [121].

The most important problem among the critical priority pathogens is their resistance to carbapenems, associated with the production of hydrolyzing them enzymes (carbapenemases), or with non-specific mechanisms such as decreasing the outer membrane permeability and overexpression of genes encoding efflux pumps [26, 37, 60]. According to Ambler classification [4], carbapenemases occurring among the strains of Gram-negative bacilli belong mainly to classes A, B and D. So far, only a few class C carbapenemases have been described, e.g., the enzyme ADC-68 in A. baumannii. Among the class A enzymes, the KPC plasmid carbapenemases (Klebsiella pneumoniae carbapenemase), mainly the variants KPC-2 and KPC-3, are of the highest clinical significance [37]. They are widespread in Enterobacterales strains, primarily in K. pneumoniae strains and increasingly in E. coli strains. The ability to produce them has also been demonstrated in Gram-negative strains of non-fermenting bacilli, i.e., P. aeruginosa, and recently in A. baumannii. The genes encoding them are located in transposons in large conjugative plasmids, which creates the possibility of their easy horizontal transfer. In the therapy of infections caused by KPC-positive strains only colistin, tigecycline and sometimes aminoglycosides can be administered. The strains producing carbapenemases of the family GES (Guiana extended-spectrum β-lactamase) are of much lower clinical importance. It is relatively rare for GES enzymes to be detected in both P. aeruginosa strains and in the bacilli of Enterobacterales, and the genes encoding them are most often located in class 1 integrons [37].

Out of the class B enzymes of the Ambler classification system (metallo-β-lactamases – MBL) [37], the enzymes of the NDM (New Delhi metallo-β-lactamase) family occur the most frequently in Gram-negative bacilli strains worldwide. Originally, enzymes of MBL type posed a therapeutic problem mainly in P. aeruginosa strains. The predominant enzymes in Europe, also in Poland, were the ones from the VIM (Verona integrated-encoded metallo-β-lactamase) family, and in the Far East, IMP (imipenemase) enzymes. The first case of the production of the NDM-1 enzyme in Enterobacterales was described in 2009, and in the following years the ability to produce NDM enzymes became widely spread, mainly in K. pneumoniae, E. coli, P. aeruginosa and other bacilli. A wide substrate range of MBL enzymes (they hydrolize all β-lactams except monobactams) in combination with the easy horizontal transfer of the genes encoding them (located in plasmids, integrons or transposons) and the lack of an inhibitor that can be utilized in therapy (they are inhibited, among others, by EDTA) make these enzymes a significant clinical problem. It is considered that the greatest threat are NDM-positive strains, whose genomes also contain genes encoding other β-lactamases (AmpC, OXA-48, VIM, KPC) and genes determining resistance to other antibiotics (aminoglycosides and fluoroquinolones). The strains producing MBL enzymes usually remain sensitive to colistin, tigecycline and fosfomycin [37].

D class carbapenemases, according to the Ambler classification system, are oxacillinases of the OXA family, the substrate range of which has been expanded, giving them the capability of hydrolysing also carbapenems. The greatest clinical importance is possessed by the enzymes of the CHDL group (carbapenem-hydrolysing class D β-lactamases) and OXA-48 β-lactamase. CHDL enzymes are the dominant mechanism responsible for carbapenem resistance in A. baumannii strains [79, 102]. In A. baumannii strains, in addition to the naturally occurring chromosomal OXA-51-like enzyme, also OXA-23-like and OXA-24-like CHDL carbapenemases are most commonly detected. In turn, the carbapenemase of OXA-48 is commonly found in the bacilli of the order Enterobacterales. The hydrolytic activity of class D carbapenemases towards carbapenems and cephalosporins is lower compared to MBL and KPC-type enzymes. However, the presence of insertion sequences (ISAba) preceding the bla_CHDL genes significantly increases the resistance of A. baumannii strains to carbapenems. A. baumannii CRAB strains, owing to the variety of other resistance mechanisms simultaneously occurring in them, are considered to be among the most dangerous pathogens, against which WHO recommends priority search for new therapeutic options.

Resistance to carbapenems has also been described among Enterobacterales strains as well as Gram-negative non-fermenting bacilli which do not produce carbapenemases. The cause of the insensitivity of the strains to carbapenems may lie in the reduction of the outer membrane permeability, due to a reduction in the abundance or changes in the conformation of the porins through which carbapenems penetrate into the cell, the so-called influx mechanism [26]. The reduced abundance or absence of OprD porins was the first reported mechanism of the resistance of P. aeruginosa to imipenem. This mechanism of resistance to carbapenems has also been described in K. pneumoniae strains (associated with OmpK35 and OmpK36 porins), E. coli (associated with OmpC and OmpF porins) and in A. baumannii (primarily CarO). Active removal of antibiotics from bacterial cells by the efflux pump systems, the so-called efflux mechanism plays an important role – in CRE strains, RND family pumps are the most important (e.g., AcrAB-TolC systems in E. coli as well as in K. pneumoniae) in CRPA strains, the MexAB-OprM and MexXY systems and the AdeABC system in CRAB strains [60]. It is often found that the resistance of Gram-negative bacilli to carbapenems includes simultaneous participation of two or three mechanisms, such as production of two different carbapenemases and/or disruption of antibiotic penetration into cells and/or active removal of drugs with the participation of efflux pump systems.

Among the pathogens from the second group according to WHO classification, the high priority kind, resistance of staphylococcal strains to methicillin and glycopeptides and resistance of enterococci to glycopeptides are the dominant problems [121]. The resistance to methicillin in staphylococci is linked to the presence of the mecA gene or its homologues, i.e., mecB, mecC or mecD, which determine the synthesis of the altered PBP-2a protein, having no affinity for β-lactam antibiotics (except for ceftaroline and ceftobiprole) [28]. The mec genes are located within a large mobile DNA fragment, i.e., SCCmec (staphylococcal cassette chromosome mec). In addition to being resistant to β-lactam, MRSA strains also typically exhibit resistance to aminoglycosides, fluoroquinolones, macrolides and lincosamins. On the other hand, they often remain sensitive to ceftaroline, ceftobiprole, linezolid, tigecycline, daptomycin, dalbavancin and rifampicin. Following the emergence of methicillin-resistant staphylococci, vancomycin became the drug of choice for the treatment of infections caused by them. Soon, however, strains with reduced susceptibility to this antibiotic (vancomycin intermediate-resistant Staphylococcus aureus – VISA), and (less frequently) vancomycin resistant strains appeared (vancomycin resistant Staphylococcus aureus – VRSA) [28]. The reasons for the reduced sensitivity of the VISA phenotype to vancomycin are not yet fully understood. Most likely, it is related to a change in the cell wall structure and a decrease in its permeability to vancomycin. In turn, the complete resistance to vancomycin, a VRSA phenotype, is conditioned by the presence of an operon containing the vanA gene. It has been demonstrated that this operon was located in the Tn1546 transposon, derived from the enterococcal conjugative plasmid. The resistance to vancomycin was first observed in enterococci. Among the VRE (vancomycin-resistant enterococci) strains responsible for hospital infections, E. faecium isolates display resistance to vancomycin significantly more often than E. faecalis [95]. The vanA gene, as well as other previously detected van genes (vanB, vanC, vanD, vanE, vanG, vanL, vanM, vanN), which determine resistance or reduced sensitivity of enterococci to vancomycin, are the ones responsible for the synthesis of the altered D-alanyl-D-alanine peptide fragment in the cell wall precursor, forming the D-alanyl-D-lactate or D-alanyl-D-serine. Thus, the target site of glycopeptide action changes, which is expressed through different levels of isolate resistance. In addition to VRE and MRSA strains, also the strains simultaneously resistant to linezolid are isolated, which may pose a serious clinical problem. The resistance of priority pathogens to fluoroquinolones results mainly from mutations in the genes encoding topoisomerase II, i.e., gyrase (in the gyrA genes) and topoisomerase IV (in the genes parC in E. coli and grlA in staphylococci), which results in the synthesis of altered subunits of these enzymes, with reduced affinity for fluoroquinolones [42]. Additionally, fluoroquinolones are substrates for many MDR pumps in priority pathogens, e.g., AdeABC in A. baumannii, MexAB-OprM in P. aeruginosa, AcrAB-TolC in Enterobacterales, NorA in S. aureus, CmeABC in Campylobacter spp. or NorM in N. gonorrhoeae [42].

Since the publication of the 2017 WHO report sounding alarm on the issue of searching for new effective drugs against dangerous pathogens, six new antibiotics from six different groups, addressing WHO priorities to various degree have been approved on the European market by the European Medicines Agency (EMA). Two of them are a new combination of carbapenems with non-β-lactam inhibitors of β-lactamases (also active against carbapenemases), from two new groups: diazabicyclooctane inhibitors (relebactam, combined with imipenem) and boronate ones (vaborbactam, combined with meropenem). The success of approving after many years new β-lactamase inhibitors from two unused before chemical groups resulted in extending the scope of the search for new inhibitors [12, 19, 39]. In turn, the search for new ways to overcome the barrier posed by the outer membrane of Gram-negative bacteria, resulted in the market authorisation of cefiderocol – siderophore cephalosporin. The use of siderophores in drug design is a novel approach to the concept of modern medication [80, 119]. Both the new combination of carbapenems with β-lactamase inhibitors and cefiderocol are active towards critical WHO pathogens to a varying degree. The next two original antibiotics comprise a novel fluoroquinolone (delafloxacin) and a new tetracycline (eravacycline), the molecules of which were designed and synthesized in order to increase efficacy and minimize the susceptibility to bacterial resistance mechanisms, typical of the older representatives of these groups. Both antibiotics are active against many WHO high priority pathogens. The last of the new antibiotics is lefamulin, the first representative of the pleuromutilin group registered for systemic use in humans. A distinctive feature of this group of antibiotics is its unique structure (meeting the WHO innovation criteria), rare occurrence of resistance and activity against pathogens of high priority according to the WHO. In turn, the strains of Gram-negative bacilli, i.e., P. aeruginosa, A. baumannii and Enterobacterales are naturally resistant to lefamulin. This article provides a review of new, wide-spectrum antibiotics active against the WHO priority pathogens, authorized for use in the European Union since 2018 (Figure 1).

Figure 1

Boronate β-lactamase inhibitors are a relatively new class of compounds with cyclic boronic acid as a pharmacophore. Vaborbactam (previously RPX7009) was dicovered under a program aimed at developing an inhibitor of serine β-lactamases, especially of the KPC type [39]. It is a monocyclic, reversible competitive inhibitor of class A enzymes according to the Ambler system (CTX-M, KPC, BKC, FRI, SME, TEM, SHV) and class C (AmpC) with which it creates covalent bonds between the boronate moiety and the catalytic serine centre. The inhibition of KPC by vaborbactam does not trigger its inactivation and is characterized by extremely slow reversibility. At the same time, vaborbactam is not active against class B enzymes (e.g., NDM, VIM) and class D (e.g., OXA-48), does not inhibit human serine enzymes and does not exhibit direct antimicrobial activity in therapeutic concentrations [15, 39, 111]. In vitro research has demonstrated that vaborbactam reduces the MIC values of carbapenems more vigorously than of the other β-lactams, hence the meropenem-vaborbactam combination was selected for further research [66].

In trials conducted on clinical isolates of Gram-negative strains of (E. coli, Enterobacter spp., K. pneumoniae, A. baumannii and P. aeruginosa), it was confirmed that vaborbactam increases the activity of meropenem against carbapenem-resistant strains of Enterobacterales but not against P. aeruginosa and A. baumannii strains [59, 97]. In large-scale surveillance programmes, the overall percentage of meropenem/vaborbactam among Enterobacterales was 99.3% and was higher than for meropenem (96.9%). In contrast, among Enterobacterales resistant to carbapenem, vaborbactam restored meropenem activity in 73.9% of the strains, and among the strains producing KPC in as much as 99.5%. Vaborbactam did not restore sensitivity to meropenem in strains producing metallo-β-lactamases and OXA-48 enzymes [88] as well as in the strains of A. baumannii, P. aeruginosa and Stenotrophomonas maltophilia [14]. Similar results were obtained by analysing the sensitivity of the collection of clinical E. coli strains resistant to carbapenems isolated around the world, in which the overall proportion of meropenem/vaborbactam-sensitive strains was 66% and was only lower than the percentage of strains sensitive to tigecycline (100%) and amikacin (74%). Sensitivity was greater among the strains of Latin American origin (88%) and lower among strains isolated in Europe (75%) and Asia (51%) [52]. Also, in the study by Zhou et al. [129], it was concluded that the proportion of K. pneumoniae strains with increased MIC values of meropenem and vaborbactam in China is higher than in other geographical areas, possibly due to the dissemination in this area of strains with defects in both major porins (OmpK35 and OmpK36) involved in the entry of carbapenems into a bacterial cell.

The growing problem of infections caused by the strains producing metallo-β-lactamases (and therefore resistant to all β-lactams, except aztreonam), combined with the absence of an effective inhibitor of these enzymes, has also resulted in attempts to combined meropenem/vaborbactam with aztreonam – a monobactam resistant to carbapenemases but destroyed by a number of serine enzymes. So far, in vitro research has demonstrated that meropenem/vaborbactam acts synergistically with aztreonam against multidrug-resistant K. pneumoniae and E. coli strains producing both NDM-type enzymes and serine β-lactamases [11, 70]. However, the effectiveness of this combination requires further research.

The hitherto observed resistance of strains to meropenem/vaborbactam was most likely associated with the production of metallo-β-lactamases [15], mutations in genes encoding porins (OmpK35, OmpK36) [24, 66, 108] responsible for drug influx and/or overexpression of AcrAB-TolC pump systems [15] involved in the efflux mechanism, less often resistance was associated with an overproduction of KPC, associated with an increase in the number of bla_KPC gene copies [108]. So far, no case of resistance associated with a mutation in the bla_KPC genes has been described.

In clinical trials it was demonstrated that meropenem/vaborbactam (administered 2 g/2 g every 8 h) is non-inferior to piperacillin/tazobactam (4 g/0.5 g every 8 h) in patients with complicated urinary tract infection (cUTI) [55]. The results of the latest clinical trial (TANGO II) further indicate that meropenemvaborbactam is superior to the best available therapy (BAT) in patients with a confirmed or suspected infection with Enterobacterales resistant to carbapenems and with multiple comorbidities such as renal failure, immunodeficiency, or prior antibiotic therapy [9, 10]. Increased cure rate, decreased mortality, and lower nephrotoxicity were observed in the patients group treated with meropenem/vaborbactam [9].

A preparation containing meropenem with vaborbactam powder for concentrate for solution for infusion (1 g/1 g) under the trade name of Vaborem has been approved on the European market since 20.11.2018 [20]. Its therapeutic indications include the treatment of complicated urinary tract infections (including pyelonephritis), complicated intra-abdominal infections, nosocomial pneumonia (including ventilator-associated pneumonia) and infections caused by Gram-negative aerobic organisms in adult patients with limited treatment options.

Relebactam (formerly MK-7655) is the second, after avibactam, non-β-lactam inhibitor of β-lactamases from the group of diazabicyclooctane derivatives, approved for treatment [20]. Relebactam is structurally similar to avibactam, however, it contains an additional piperidine ring which ensures a positive charge at physiological pH for the molecule, crucial for reducing its susceptibility to active removal by pump systems in the phenomenon of efflux. Biochemical analyses have demonstrated that relebactam is an inhibitor of class A (of KPC type) and class C enzymes (AmpC type, e.g., AmpC Pseudomonas-derived cephalosporinase-3, PDC-3) and in addition (similarly to imipenem), it is not a substrate for the MDR pump systems in P. aeruginosa [7, 12, 124]. The lack of susceptibility of the imipenem/relebactam combination to being removed from bacterial cells by the P. aeruginosa MDR pump systems and the activity of relebactam against AmpC enzymes resulted in this combination being selected for further research. In vitro research has confirmed that relebactam actually increases the activity of imipenem against Enterobacterales strains, whose carbapenem resistance was associated with the production of KPC enzymes or in which the production of AmpC or ESBL β-lactamases was observed combined with the reduced permeability of the outer membrane (mutations in OmpK36). As was also observed in the P. aeruginosa OprD-deficient strains and (at higher concentrations of the drug) against P. aeruginosa MDR strains. Similarly to vaborbactam, relebactam is not an inhibitor of metallo-β-lactamases (NDM, IMP, VIM) or class D enzymes (OXA), neither does it display direct antibacterial activity [36, 40, 63]. The synergistic interaction of imipenem/relebactam with amikacin and colistin against imipenem-resistant P. aeruginosa strains has also been described under in vitro conditions [6].

In many large surveillance studies, the susceptibility to imipenem/relebactam of clinical strains isolated from patients with lower respiratory tract infections, urinary tracts infections and intra-abdominal infections has been tested [53, 54, 64, 65]. It has been demonstrated that in the presence of relebactam, imipenem concentrations able to prevent the growth of most clinical Enterobacterales strains (including carbapenem-resistant strains) were reduced, although the MIC values of imipenem for Serratia marcescens strains are usually higher than for other species. The percentage of imipenem/relebactam-sensitive strains of E. coli, Klebsiella spp., Citrobacter spp., and Enterobacter spp. reached over 95%. However, among S. marcescens the percentage of susceptible strains was lower. Imipenem/relebactam is also effective against P. aeruginosa strains (the percentage of susceptible strains reaching over 90%), with the exception of strains producing β-lactamases of class B or D. Among the strains resistant to carbapenems, 42–66% of Enterobacterales strains and 74–78% of P. aeruginosa strains remain sensitive to imipenem/relebactam. Relebactam, however, does not increase the activity of imipenem against Acinetobacter spp. [53, 54, 64, 65, 115].

The observed resistance of Gram-negative bacilli to imipenem/relebactam stems mainly from the production of metalloenzymes. In P. aeruginosa strains, it may also be due to the ability to produce class A carbapenemases from the GES family and overexpression of genes encoding PDC enzymes in combination with the loss of the OprD porin proteins [65, 124]. In Enterobacterales, in turn, resistance may be caused by the production of oxacillinases with carbapenemase activity, e.g., OXA-48, as well as mutations in the genes encoding porin proteins and a decrease in the abundance of these porins (OmpK35, OmpK36, OmpC and OmpF), thereby limiting imipenem/relebactam penetration into bacterial cells [30, 51].

Clinical studies have demonstrated that imipenem (administered at a dose of 500 mg every 6 hours) in combination with relebactam (125 mg or 250 mg every 6 hours) is a well-tolerated treatment and is non-inferior than imipenem used alone in the group of patients with complicated intra-abdominal infections and complicated urinary tract infections [67, 101]. Imipenem/relebactam was also characterized by comparable efficacy and lower nephrotoxicity than the use of imipenem and colistin in the combination therapy of infections caused by imipenem-resistant Gram-negative bacilli [78]. On the basis of the obtained results, a preparation containing imipenem/cilastatin (where cilastatin is an inhibitor of dehydrogenase I, a kidney enzyme which inactivates imipenem) with relebactam (Recarbrio 500 mg/500 mg + 250 mg, powder for solution for infusion) was approved by EMA on 13.02.2020 for the treatment of nosocomial pneumonia (also associated with mechanical ventilation) and in the treatment of Gram-negative bacterial infections in adults with limited therapeutic options [20].

Cefiderocol is a representative of a new group of antibiotics – siderophore cephalosporins [80]. Siderophores are a structurally diverse group of small molecules (150–2000 Da) with chelating properties and high iron affinity [119]. Iron is an essential element for the functioning of many enzymes, and therefore also for microorganisms. However, its acquisition by bacteria is hampered due to the low solubility of iron at physiological pH under aerobic conditions, in which easily bioavailable Fe (II) ions are oxidized to Fe (III), causing the formation of insoluble ferric oxyhydroxides. In addition, host defence mechanisms cause further reduction in iron accessibility at the infection site due to the secretion of their own iron-binding proteins, such as lipocalin 2, also called siderocalin, or neutrophil gelatinase associated lipocalin (NGAL). In order to obtain the sufficient amount of iron, the bacteria produce and secrete siderophores into the extracellular environment, whose task is to bind its ions and transport them inside the cell. More than 500 different siderophores have been identified so far. Characteristic structural elements in their molecules are iron chelating functional groups (hydroxamates, catechols, carboxylates, phenolate moieties or combinations thereof) attached to a linear or cyclic scaffold forming a hexadentate structure. After binding an iron ion, the resulting complex (ferrisiderophore) is absorbed in the mode of active transport. In Gram-negative bacteria, the entire complex crosses both the outer and inner membranes, and the bounded iron is released in the cytoplasm. Alternatively, iron may be released in the periplasmic space [119].

The first attempts to use siderophores to transport antibiotics inside the cell were made already in the 1970s. In the 20th century, this strategy was called the “trojan horse approach” [80]. Cefiderocol (formerly S-649266, GSK2696266) is the first antibiotic of this type introduced into healthcare. It is used in the form of cefiderocol sulphate tosylate. It is a catechol siderophore cephalosporin, which was selected from other derivatives of a similar structure, in the course of a program aimed at finding a new antibiotic active against carbapenem-resistant strains [5]. Cefiderocol has a structural similarity to cefepime, such as the presence of a pyrrolidinium group on the C-3 side chain, increasing antimicrobial activity and stability against β-lactamases, and a carboxypropanoxyimino group on the C-7 side chain, which improves the transport of cefiderocol across the outer membrane. Additionally, cefiderocol also has a chlorocatechol group at the end of the C-3 side chain, which is responsible for siderophoric activity [96].

The principal mechanism of action of cefiderocol is the inhibition of cell wall synthesis by binding to PBPs (mainly PBP-3). Cefiderocol, after binding iron, reaches the periplasmic space through active transport, in which, inter alia, CirA and Fiu transporters in E. coli and PiuA in P. aeruginosa are involved. This transport mechanism eliminates the problem of resistance associated with reducing the number of porins in the outer membrane or the overexpression of the MDR pumps, which are responsible for the phenomenon of efflux [96]. Furthermore, a cefiderocol molecule is resistant to a wide range of β-lactamases, both serine (KPC-3, OXA-23, AmpC) and metallo-β-lactamases (IMP-1, VIM-2) [46, 47]. However, this resistance may arise due to mutations within the genes encoding either PBPs, or a protein related to the regulation of iron ion uptake or siderophores transport protein, as well as the production of β-lactamases capable of hydrolysing cefiderocol (NDM, PER type) and overexpression of native bacterial siderophores [58, 69, 96].

Cefiderocol is highly active against a broad spectrum of Gram-negative bacteria, both representatives of Enterobacterales (Enterobacter spp., Klebsiella spp., Proteus spp., S. marcescens, Shigella flexneri, Salmonella spp., Yersinia spp.) as well as non-fermenting bacilli (Acinetobacter spp., Pseudomonas spp., Burkholderia spp., S. maltophilia) and Vibrio spp. [48]. However, it displays no activity against aerobic Gram-positive bacteria (Staphylococcus spp., Enterococcus spp.) as well as anaerobic bacteria. Cefiderocol is also active against strains producing various β-lactamases, such as the KPC, VIM, NDM and OXA-48 types in Enterobacterales, or the VIM, IMP, NDM and GES types in P. aeruginosa or CHDL enzymes from the OXA-23, OXA-24/40 and OXA-58 groups in A. baumannii [48]. The sensitivity of the clinical strains of Enterobacterales and non-fermenting bacilli has been analysed in several international surveillance studies [27, 35, 106]. So far, the percentages of cefiderocol-sensitive strains isolated from patients suffering from, e.g., nosocomial pneumonia, bloodstream infection, complicated intra-abdominal infections and complicated urinary tract infections are at a level > 95%, irrespective of the geographic region. A lower percentage was observed only in the case of K. pneumoniae strains (88%).

In clinical studies it has been demonstrated that cefiderocol (administered 2 g every 8 hours) is non-inferior to imipenem (1 g every 8 hours) in the treatment of complicated urinary tract infections (cUTI) (APEKS-cUTI study) [89], and non-inferior to meropenem (2 g every 8 hours) in the treatment of nosocomial pneumonia (also linked to mechanical ventilation) (APEKS-NP) [122]. It has also been demonstrated that cefiderocol has similar clinical and microbiological efficacy in the treatment of critically ill patients infected with carbapenem-resistant Gram-negative bacilli, compared to the best available therapy (BAT), although in the group treated with cefiderocol, higher mortality has also been reported, mainly in patients with infections caused by Acinetobacter spp. [8]. Cefiderocol was approved by EMA on 23.04.2020 under the name of Fetcroja 1 g, powder for concentrate for solution for infusion. Its current therapeutic indications include the treatment of infections caused by aerobic Gram-negative bacteria in adults, with limited therapeutic options [20].

Delafloxacin (formerly WQ-3034, ABT-492) is a new 4 th generation fluoroquinolone and the first anionic compound in this group [57, 112]. Its molecule is distinguished primarily by the absence of the basic group in the C7 position (which ensures acidic properties), the presence of chlorine in the C8 position, which serves as an electron-withdrawing group on the aromatic ring (which increases the polarity of the compound and improves its activity and stability) and the presence of a voluminous heteroaromatic substituent in the N1 position, whereby the surface area of delafloxacin is much larger than that of other fluoroquinolones. As a result, delafloxacin exists in an anionic form at neutral pH and in a neutral form in an acidic medium. This contributes to its increased activity under low pH conditions, while the activity of other fluoroquinolones decreases as the pH drops.

The mechanism of action of delafloxacin, like all fluoroquinolones, consists in inhibiting gyrase and topoisomerase IV [81]. However, it has been shown to act with comparable potency on both of these topoisomerases in both E. coli and S. aureus. Whereas the remaining fluoroquinolones are more active against topoisomerase IV in Gram-positive bacteria and against gyrase in Gram-negative bacteria. The inhibition of gyrase activity is a more effective way of inhibiting DNA replication due to the involvement of this enzyme at an earlier stage (removal of positive super-coils before the replication forks) than in the case of topoisomerase IV, which operates behind the replication forks (DNA decatenation and chromosomal separation). Thus, the stronger interaction of delafloxacin with gyrase in Gram-positive bacteria (in comparison to the remaining fluoroquinolones) contributes to the increased activity of this new fluoroquinolone against these bacteria.

Additionally, the similar affinity to both topoisomerases suggested that delafloxacin, compared to other fluoroquinolones, should predispose strains to developing resistance to a lesser degree due to the necessity to create mutations simultaneously in both genes encoding these topoisomerases. In vitro studies have demonstrated a lower potential for the selection of resistant, spontaneous mutants of S. aureus MRSA strains than in the case of other fluoroquinolones [92]. The mutant prevention concentration (MPC) values of delafloxacin were 8 to 32 times lower than the MPC values of moxifloxacin, levofloxacin and ciprofloxacin, and additionally, in the obtained mutants, compared to the parent strain, a decrease in viability was observed. The sensitivity analysis of the strains isolated from patients treated with delafloxacin during clinical examinations confirmed that delafloxacin retained high activity also against the strains with mutations in quinolone resistance-determining regions (QRDR) [72]. Delafloxacin MIC values were not significantly increased (i.e., MIC > 0.5 mg/l) until there were simultaneous double mutations in both gyrA and parC genes.

As anticipated, in in vitro studies, delafloxacin demonstrated more significant activity than trovafloxacin, levofloxacin and ciprofloxacin against sensitive and quinolone-resistant strains of Gram-positive bacteria (Staphylococcus spp., Enterococcus spp., Streptococcus spp., Listeria monocytogenes), fastidious Gram-negative bacteria (H. influenzae, Moraxella catarrhalis, N. gonorrhoeae, Legionella pneumophila) and H. pylori. Its effectiveness against Enterobacterales and P. aeruginosa strains was comparable with other fluoroquinolones, and its effectiveness against Chlamydia spp. was comparable with trovafloxacin and higher than levofloxacin [3, 29, 81]. Under in vitro conditions, delafloxacin is also effective against Mycoplasma pneumoniae, Mycoplasma fermentans, Mycoplasma hominis and Ureaplasma spp. [113]. It also shows activity against the biofilm formed by S. aureus MSSA and MRSA, with the ability to penetrate into its interior reaching 52% (depending on the proportion of polysaccharides in the matrix) and increased effectiveness against biofilms of lower pH [100]. It has been demonstrated that delafloxacin usually has a bactericidal effect against Streptococcus pneumoniae, H. influenzae and M. catarrhalis strains [34].

The high activity of delafloxacin against staphylococci (also MRSA) and streptococci, the strains most often causing acute bacterial skin and skin structure infections (ABSSSI), combined with its high activity in the acidic environment (typical for the skin), directed further research on the application of delafloxacin to this disease. The recently published results of the surveillance studies of the sensitivity of 11,866 strains isolated over the years 2014–2019 in the USA and Europe from patients with ABSSSI confirmed the high activity of delafloxacin [99]. The most frequently isolated strains were S. aureus MSSA (~ 30.6%), E. coli (11%), Streptococcus spp. (10%) and S. aureus MRSA (7.2%). The susceptibility to this antibiotic was confirmed for 98.7% of MSSA strains, 98.4% of Streptococcus spp. strains, 58% of E. coli strains and 65.6% of MRSA strains.

In clinical trials, delafloxacin turned out to be a drug with linear pharmacokinetics, minimal accumulation and was well-tolerated – gastrointestinal side effects were observed only at single oral doses > 1200 mg or multiple doses > 800 mg [44]. An oral dose of 450 mg and an intravenous dose of 300 mg proved to have comparable effects, providing an option of changing the route of administration during the therapy [43].

In randomized clinical trials in patients with complicated infections of the skin and subcutaneous tissue (abscesses and wound infections after surgery, trauma, burns and bites) intravenous delafloxacin (300 mg twice daily) was as effective as tigecycline (50 mg twice daily) [83], vancomycin with aztreonam (15 mg/kg + 2 g twice daily) [91] and linezolid (600 mg twice daily) [56] and more effective than vancomycin (15 mg/kg of body mass twice daily) [56]. Efficacy not worse than that of vancomycin with aztreonam has also been demonstrated for delafloxacin with the dosage pattern of 300 mg intravenously (twice daily) for 2 days and then 450 mg orally [82]. The same dosage pattern for delafloxacin was non-inferior than moxifloxacin (400 mg intravenously followed by 400 mg orally once daily) in the treatment of community-acquired bacterial pneumonia (CABP), also pneumonia caused by atypical pathogens (M. pneumoniae, Chlamydia pneumoniae, L. pneumophila) [45]. Delafloxacin showed at least 16-fold greater activity than moxifloxacin against Gram-positive bacteria and fastidious Gram-negative bacilli, and also retained activity against resistant strains, e.g., S. pneumoniae MDR, Haemophilus spp. producing β-lactamases and macro-lide-resistant strains, as well as S. aureus MRSA and fluoroquinolone-resistant strains [71, 73].

However, a single 900 mg dose of delafloxacin turned out to be an ineffective form of treating uncomplicated gonorrhoea – many treatments for N. gonorrhoeae infections with MIC values below 0.008 mg/l were unsuccessful, indicating the need to modify the dosing regimen [41].

In the European Union, delafloxacin was approved on 16.12.2019 for the treatment of acute bacterial skin and skin structure infections (ABSSSI) and community acquired pneumonia (CABP) in adults, when the application of other antibacterial agents commonly recommended for the initial treatment of these infections is considered to be inappropriate. Both the oral (450 mg tablets) and intravenous (300 mg) forms were registered under the trade name Quofenix [20].

Tetracyclines are natural or semi-synthetic compounds with amphoteric properties, containing four fused carbocyclic rings (including an aromatic one) in their molecule [31]. Their mechanism of action consists in inhibiting protein synthesis by binding to the 30S ribosome subunit. All compounds from this group are characterized by a broad spectrum of activity, both against Gram-positive and Gram-negative bacteria, mycoplasmas, chlamydia, rickettsiae and some protozoa. The differences between individual representatives concern the pharmacokinetic properties and potency. The first tetracycline antibiotics (produced by various species of actinomycetes – Streptomyces) were introduced into medicine at the turn of the 1940s and 1950s. Unfortunately, their intensive use in medicine led to the rapid emergence and spread of resistance associated with the presence of tet genes, encoding membrane pump proteins involved in the phenomenon of the so-called efflux (e.g., tet(A), tet(B), tet(K), tet(L) genes), ribosome protective proteins (e.g., tet(M), tet(O), tet(P), tet(S)) or tetracycline-inactivating enzymes, i.e., the tet(X) gene.

However, the growing drug resistance of microorganisms to antibiotics from other groups contributed to the renewed interest in tetracyclines. Determining the structure of the tetracycline-30S ribosome co-crystal demonstrated that the binding to the ribosome does not involve only the region around the C5–C9 carbon atoms [13]. Therefore, attempts were made to introduce structural modifications at C7 and C9 atoms, which resulted in the market launch of semi-synthetic tetracyclines (minocycline, tigecycline, omadacycline). The new tetracyclines are characterized by increased antibacterial activity and lower susceptibility to bacterial resistance mechanisms [31]. However, the number of the chemical modifications of natural tetracyclines and thus the possibility of obtaining new, semi-synthetic derivatives is limited. It was only the de novo synthesis of subsequent compounds containing the tetracycline skeleton that significantly increased the number of new compounds from this group [107].

Eravacycline (formerly TP-434) is the first fully synthetic tetracycline introduced into medicine [20]. Its molecule is characterized by the presence of fluorine at the C7 carbon atom and the pyrrolidinoacetamido group at the C9 carbon of the tetracycline core and is also active against tetracycline-resistant strains with the tet(M) and tet(Q) genes (responsible for blocking the tetracycline binding site to the ribosome) and the genes tet(A), tet(B) and tet(K) (responsible for the synthesis of membrane proteins of MDR pumps) [33, 123]. The spectrum of eravacycline activity includes strains of S. aureus (also linezolid resistant MRSA and MRSA), Streptococcus spp., Enterococcus spp. (also VRE), E. coli, K. pneumoniae, Klebsiella oxytoca, Enterobacter cloacae, Enterobacter aerogenes, Citrobacter freundii, Morganella morganii, Proteus mirabilis, Proteus vulgaris, Providencia stuartii, S. marcescens, Salmonella spp., Shigella spp., A. baumannii, Acinetobacter lwoffii, S. maltophilia, Legionella pneumophila, H. influenzae, M. catarrhalis, N. gonorrhoeae, Bacteroides fragilis, Bacteroides vulgatus, Bacteroides thetaiotaomicron, Bacteroides ovatus, Prevotella spp., Clostridioides difficile, and Clostridium perfringens. Eravacycline maintains its effectiveness also against strains resistant to fluoroquinolones, aminoglycosides, third-generation cephalosporins, carbapenems, polymyxins and MDR strains, and its effectiveness is usually 2–4 times higher than or equal to tigecycline, both against Gram-positive and Gram-negative bacteria [1, 23, 50, 62, 103, 110, 125, 127]. It has also been shown that under in vitro conditions eravacycline acts synergistically with colistin against A. baumannii strains, also against strains resistant to carbapenems and colistin [84]. Nevertheless, the strains of P. aeruginosa and Burkholderia cenocepacia are resistant to eravacycline [110].

Moreover, it has been demonstrated that eravacycline is effective against the biofilm of the uropathogenic E. coli strain to a degree comparable to that of gentamicin and levofloxacin, and to a greater degree than in the case of colistin and meropenem [32]. However, eravacycline has not been shown to be effective against the biofilm formed by S. aureus strains isolated from periprosthetic-joint infections [130].

Recently published results of large surveillance studies have confirmed the high effectiveness of eravacycline against a wide spectrum of clinical strains [76, 77]. The percentage shares of susceptible strains were respectively: 97.6% for S. aureus (95.5% for MRSA and 99.8% for MSSA), 84.6% for S. epidermidis, 89.9% for S. haemolyticus, 99.4% for E. faecalis (98.3% for VR strains, 99.5% for VS), 97.7% for E. faecium (96.1% for VR strains and 98.9% for VS) and 92.6% for Enterobacterales (82% for MDR strains). In turn, for A. baumannii strains for which no MIC breakpoints have been defined, the MIC₅₀/MIC₉₀ values reached 0.5 mg/l and 1 mg/l, respectively (for MDR strains the MIC₉₀ reached 2 mg/l).

However, the clinical effectiveness of eravacycline may be threatened in the future by the increasing resistance arising from the antibiotic-removal mechanism (efflux), enzymatic degradation of this antibiotic and modification of its binding site in the ribosome. So far, it has been demonstrated that in S. aureus (both MRSA and MSSA), E. faecalis and S. agalactiae strains, the MIC values of eravacycline (similar to tigecyclines) showed significant decreases in the presence of MDR pump inhibitors such as carbonyl cyanide 3-chlorophenyl-hydrazone (CCCP) and L-phenylalanine-L-arginine-β-naphthylamide (PAβN) [61, 118, 127]. Additionally, research describes clinical strains of A. baumannii resistant to eravacycline with overexpression of the AdeABC pump system [98], K. pneumoniae with over-expression of OqxAB and MacAB-TolC pumps [128] as well as K. pneumoniae PDR (pan-drug resistant) strains isolated from farm animals in China with the expression of a new RND efflux pump, of the MexCD-OprJ type, called TMexCD1-TOprJ1, responsible for the resistance, among others to all tetracycline antibiotics [68]. Since the genes encoding it are located in the plasmid, their spread among zoonotic strains of K. pneumoniae may pose a serious clinical threat in the future. Another, equally important threat to the effectiveness of applying eravacycline in therapy is the spread of tet(X) genes located in plasmids and transposons. Several variants of these genes have recently been described in China in A. baumannii strains (environmental and clinical) [16, 116] and in Enterobacterales strains isolated from the stool samples of healthy adults in Singapore [18]. They are also widespread among E. coli strains isolated from animals (pigs, chickens, migratory birds) and soil [17, 109], and recently the presence of these genes has also been confirmed in Proteus sp. isolated from retail pork [38]. Resistance to eravacycline may also result from mutations in the genes encoding the proteins of the 30S ribosome subunit, as already described in S. aureus and S. agalactiae strains [61, 117].

Clinical studies have shown that when it comes to the treatment of complicated intra-abdominal infections, eravacycline (administered 1 mg/kg every 12 hours) is well tolerated and safe, as well as non-inferior to meropenem (administered 1 g every 8 hours) and non-inferior to ertapenem (1 g every 24 hours) [104, 105]. Based on the results obtained, eravacycline was approved by the EMA for use against this nosological unit on 20.09.2018 and registered under the trade name Xerava 50 mg, powder for concentrate for solution for infusion [20].

Lefamulin is a new antibiotic from the group of pleuromutilins – derivatives of naturally occurring pleuromutilin, isolated in the 1950s from the fungus Pleurotus mutilus (presently, Clitophilus scyphoides) [86]. The first semi-synthetic derivatives of pleuromutilin (tiamulin and valnemulin) were approved for veterinary use in 1979 and 1999, respectively. However, these drugs were used only in the treatment of pulmonary and intestinal infections in animals but not in the production of food of animal origin as growth promoters or for enhancement of feed efficiency (unlike, among others, tetracyclines, streptogramins or sulphonamides). This may explain the low prevalence of bacterial resistance to pleuromutilins. Retapamulin was the first antibiotic of this class approved for human use, but it was only available as an ointment for topical application for a short-term treatment of superficial skin infections (impetigo, minor lacerations, abrasions, or sutured wounds). Lefamulin (formerly BC-3781) is the second pleuromutilin approved for human use, but the first to be approved for systemic use, both in the intravenous and oral forms [86].

Chemically, pleuromutilins are diterpenoids containing in their molecule a 14-carbon tricyclic scaffold (essential for antimicrobial activity) and a glycolic ester moiety forming a side chain at the C14 position, various modifications of which correspond, among others, to different pharmacodynamic properties of derivatives [126]. The mechanism of action of this group of antibiotics consists in inhibiting protein synthesis by binding to the 50S ribosome subunit at the peptidyl transferase centre (PTC), in the middle of the domain V of the 23S rRNA molecule. A tricyclic core that forms hydrogen bonds with the nucleotides in the pocket near the A-site of the ribosome is responsible for the attachment of pleuromutilins to the ribosome, while the side chain at position C14 extendes towards the P-site, hindering the movement of the 3′ tRNA end towards the P site. A lefamulin molecule features a stronger bond to the ribosome than other pleuromutilins due to the presence of the 2-(4-amino-2-hydroxycyclohexyl) sulfanylacetate side chain at the C14 position, the amino group of which allows the formation of another hydrogen bond. Additionally, the presence of hydroxyl and primary amine groups in this chain increases the solubility of lefamulin in water [21].

Under in vitro conditions, lefamulin is effective against most Gram-positive cocci (S. aureus MSSA and MRSA, S. epidermidis, vancomycin-resistant E. faecium, S. pneumoniae (also resistant to penicillin, macrolides and MDR strains), S. pyogenes, S. agalactiae, Streptococcus spp. groups C and G), and some Gram-negative bacteria (H. influenzae, Haemophilus parainfluenzae, L. pneumophila, M. catarrhalis, N. gonorrhoeae) and atypical pathogens (M. pneumoniae, Ch. pneumoniae). However, it is not active against Gram-negative non-fermenting bacilli and Enterobacterales [49, 74, 85, 93, 94, 114].

Surveillance studies have confirmed that lefamulin is exceedingly active against pathogens causing community-acquired pneumonia, isolated from patients worldwide (SENTRY Antimicrobial Surveillance Program) [85, 87]. The overall percentage share of strains with the MIC values for lefamulin below 1 mg/l (breakpoint for S. pneumoniae) was 99.2%, including 100% for S. pneumoniae and M. catarrhalis strains, 99.8% for S. aureus, 93.8% for H. influenzae and 88.1% for E. faecium. Simultaneously, no cross-resistance was detected between lefamulin and β-lactams, fluoroquinolones and macrolides.

Resistance to lefamulin in the strains of usually sensitive bacterial species may be a result of mutations in the V domain 23S rRNA, e.g., due to the nucleotide methylation at position 2503 influenced by Cfr methyltransferase [75]. The Cfr enzyme provides cross resistance to oxazolidinones, lincosamides, phenicols and streptogramins. Furthermore, the acquisition of resistance may result from mutations in the genes encoding the ribosomal protein L3 (rplC) and L4 (rplD), which modifies the structure of PTC and disturbs lefamulin attachment to it. It has been demonstrated that the resistance may also be related to the protection of the lefamulin target binding site by membrane pump proteins of the ABC-F subfamily encoded by the vga(A), vga(B), vga(E) and Isa(E) genes. ABC-F proteins may induce cross-resistance to lincosamides and streptogramins A [75]. It has also been demonstrated that the inactivation of the MtrCDE pump (but not MacAB and NorM) in N. gonorrhoeae reduces significantly (at least 4-fold) the MIC values of lefamulin [49].

In clinical trials it has been demonstrated that lefamulin (administered 150 mg intravenously every 12 hours for 3 days, followed by 600 mg orally every 12 hours) is non-inferior as moxifloxacin (400 mg administered intravenously every 24 hours for 3 days, followed by 400 mg orally every 24 hours) in the treatment of community-acquired pneumonia [22]. Comparable results were obtained when lefamulin and moxifloxacin were administered orally exclusively [2]. At the same time, in the treatment of acute bacterial skin and skin structure infections caused by Gram-positive pathogens, in the group of patients treated with lefamulin (100 or 150 mg intravenously every 12 hours), the percentage of clinical successes was comparable to the group of patients treated with vancomycin (1 g intravenously every 12 hours) [90]. Currently, the intravenous (150 mg injections every 12 hours) and oral (600 mg tablets) forms of lefamulin, under the trade name Xenleta, were approved for use on 27.07.2020 only in the treatment of community-acquired pneumonia in adults [20].

Six new, broad-spectrum antibacterial drugs have been registered on the European market since 2018, which are, according to WHO, active against the strains classified as pathogens with a critical and high priority need of search for new drugs. Two of them are new combinations of β-lactams with non-β-lactam inhibitors of β-lactamases (meropenem with vaborbactam and imipenem/cilastatin with relebactam), one is a representative of a new group of siderophore antibiotics (cefiderocol), the next two are new derivatives of known groups of antibiotics: fluoroquinolones (delafloxacin) and tetracyclines (eravacycline). Moreover, EMA approved lefamulin as the last of the new drugs in July 2020, i.e., the first representative of a new group of drugs – pleuromutilin, intended for systemic use in humans. Lefamulin, unlike the aforementioned new drugs, is not active against Gram-negative non-fermenting bacilli and Enterobacterales, which are naturally resistant to it. According to the criteria adopted by the WHO, two of the 6 new antibacterial drugs (delafloxacin and lefamulin) were registered as effective against high priority pathogens (vancomycin-resistant enterococci, MRSA), and the remaining four (meropenem/vaborbactam, imipenem/cilastatin/relebactam, eravacycline and cefiderocol) as active against critical pathogens (carbapenem-resistant Enterobacterales), while only one (cefiderocol) displays high effectiveness also against carbapenem-resistant strains of P. aeruginosa and A. baumannii. The WHO innovation criterion is fully met only by meropenem/vaborbactam (in the new chemical class category) and partially by lefamulin, with the reservation that the representatives of this group (pleuromutilins) have already been used in veterinary medicine and for topical treatment in humans. On the other hand, the innovation criterion in the category of the lack of cross-resistance is potentially met by meropenem/vaborbactam and cefiderocol.

Two of the approved drugs are available in both intravenous and oral formulations (delafloxacin and lefamulin). Owing to new registrations, there has been an increase in the number of therapeutic options in the treatment of complicated urinary tract infections (meropenem/vaborbactam, cefiderocol), complicated intra-abdominal infections (meropenem/vaborbactam, eravacycline), nosocomial pneumonia, including those associated with mechanical ventilation (meropenem/vaborbactam, cilastatin/relebactam), acute bacterial skin and skin structure infections (delafloxacin) and community-acquired pneumonia (lefamulin). According to the recent WHO report on new antibiotics, the number of new therapeutic options available for the treatment of infections caused by high and critical priority pathogens, as well as PDR (pan-drug resistant) and XDR (extensively drug resistant) pathogens is still insufficient [120]. Recently, however, a case of therapeutic success has been reported in the treatment of Achromobacter sp. PDR infection in a 10-year-old female patient with cystic fibrosis after applying the combination of cefiderocol, meropenem with vaborbactam and phage therapy (Ax2CJ45ϕ2). The treatment was well tolerated and led to the eradication of Achromobacter sp. [25]. It is thus possible that, owing to the combination therapy, the new antibiotics will also prove effective against PDR and XDR pathogens, which will enhance the prospects of their application in healthcare.