What do We Know so Far about Ges Carbapenemases, and What Threat do They Pose?

The carbapenemase active site of GES-2 exhibits a similar distinguishing pattern to that observed in GES-1, with serine (Ser70), lysine (Lys73) and glutamate (Glu166) all playing a comparable role. However, a critical mutation resulted in the conversion of gly-cine-to-asparagine (Asp170), which is responsible for increasing the enzyme activity (Poirel et al. 2001).

A characteristic feature of this carbapenemase is that it has a canonical asparagine at position 170, unlike GES-4, -5, and -6, which have a Gly170Ser substitution at this position. In this enzyme, a hydrolytic water molecule positions itself between Ser70 and Glu166 and is also bound to Asn170. The presence of GES-2, like GES-4, -5, and -6, causes a decrease in bacterial susceptibility to imipenem. Research conducted by Frase et al. (2011) showed that blocking this carbapenemase with tazobactam (at a concentration of 4 μg/ml) changed the MIC (minimum inhibitory concentration) for piperacillin from > = 128 μg/ml (resistance) to 1 μg/ml. Based on this, it can be concluded that tazobactam, in combination with carbapenems, can be used in therapy against bacteria with GES-2 resistance. The dissociation constant for the noncovalent complex of GES-2 and tazobactam was in the nanomolar range, indicating the high affinity of tazobactam for GES-2. Additionally, studies have shown that the mentioned inhibitor has a rapid onset of enzyme inhibition (Frase et al. 2011).

The GES-4 carbapenemase active site exhibits a similar function to that of GES-1, with the presence of serine (Ser70), lysine (Lys73) and glutamate (Glu166). However, critical mutations have occurred, resulting in the conversion of glycine to serine (Ser170), which is responsible for increasing the enzyme activity. Additionally, the conversion of alanine to valine (Val173) affects the substrate specificity and efficiency of the enzyme (Wachino et al. 2004; Barlow and Tenover 2024).

Research conducted by Vourli et al. (2006) showed that after exposure K. pneumoniae 78–01 strain with GES-4 and SHV-5 resistance to clavulanic acid in combination with imipenem or ceftazidime, the susceptibility to these antibiotics was partially restored. The gene encoding GES-4 (blaGES-4), in the form of genomic cassettes, is located in the variable regions of class 1 integrons, which are carried by plasmids, and this enables horizontal transfer of this resistance between bacteria (Bebrone et al. 2013).

The active center of the GES enzyme 5, a car-bapenemase, is structured similarly to other class A β-lactamases. It contains a catalytic serine residue in its active site, crucial for its hydrolytic activity. This serine is part of a conserved sequence motif (Ser70-X-X-Lys73) that plays an essential role in the enzyme’s ability to hydrolyze β-lactam antibiotics (Smith et al. 2012).

The study performed by Kotsakis et al. (2010) showed that this enzyme has the highest carbapenemase activity, which is associated with the presence of serine at position 170. The Gly170Ser substitution increases the ability to hydrolyze cefoxitin and imipenem but also causes a decrease in activity towards ceftazidime and aztreonam. The presence of serine at position 170 changes the structure of the enzyme, namely in the Ω loop and results in improved catalytic properties of the enzyme against carbapenems and cephamycin; it also increases the resistance to β-lactamase inhibitors (Poirel et al. 2018).

IR-GES-5 refers to an integron-associated GES-5 (Guiana Extended Spectrum) β-lactamase enzyme. The “I. R.” typically stands for Integron-encoded Resistance, indicating that the gene encoding this enzyme is located within an integron, a genetic element in the bacterial genome. The association of GES-5 with IR-GES-5 enhances its ability to spread rapidly across different bacterial species, making it a concern in the treatment of bacterial infections. The active center of the IR-GES-5 enzyme, like other GES-type β-lactamases, possesses a catalytic serine residue (Ser70). The structure of the active site allows GES-5 to bind and hydrolyze carbapenems, which are often resistant to degradation by other β-lactamases (Labuschagne et al. 2008).

The active center of the GES enzyme 6 (GES-6), like other GES-type enzymes, features a serine-based mechanism typical of class A β-lactamases. The key residues in the active site are Lys and Ser at the 104 and 170 positions, respectively. The active center also includes other residues, such as Lys73, Ser130, Glu166, and Asn170, which are involved in substrate binding, catalysis, and stabilization of the transition state (Kotsakis et al. 2010).

Compared to GES-1, GES-6 has more significant activity against carbapenems and ceftolozane and reduced susceptibility to β-lactamase inhibitors (except avibactam). It is worth noting that when ceftolozane was combined with tazobactam, the MIC decreased slightly compared to the value for ceftolozane without the inhibitor, demonstrating the reduced effectiveness of the inhibitors. However, the activity of GES-6 towards imipenem was higher than in GES-1, which confirmed the involvement of Ser170 in the higher activity of the enzyme. It should be noted that the substrate profile of GES-6, in some sense, reflects MBLs with activity against carbapenems and some resistance to inhibitors (Poirel et al. 2018).

Botelho et al. (2015) showed that the blaGES-6 gene in the P. aeruginosa strain is accompanied by the aacA7 gene encoding aminoglycoside acetyltransferase type 1, conferring resistance to amikacin, netilmicin, and tobramycin.

This enzyme was first discovered in 2008 in France in Acinetobacter baumannii (Moubareck et al. 2009). Substitution of the glycine at position 243 in GES-11 was associated with increased activity toward aztre-onam, as had been observed for GES-9. GES-11 did not have a substitution of the Gly170 residue, resulting in increased hydrolysis of imipenem as in GES-2, GES-4, GES-5, and GES-6. Research conducted by Moubareck et al. (2009) showed that expression of the blaGES-11 gene in porin-deficient cells may lead to resistance to imipenem. The active site of GES-11 is similar to other serine carbapenemases, including serine at position 70 (Ser70), lysine at position 73 (Lys73), glutamate at position 166 (Glu166) and glycine at position 170 (Gly170). The lack of substitution of the Gly170 residue increases the hydrolytic properties of this enzyme. GES-11 has not been fully classified, and research is still ongoing on whether it belongs to β-lactamases or carbapenemases (Moubareck et al. 2009).

The active site of GES-14, considering key amino acid positions, is similar to other carbapenemase-active GES variants: Ser 70, Lys 73, Glu 166, Gly 170. The exact sequence of the amino acids surrounding these residues defines the enzyme’s active site. Unlike GES-11 carbapenemase, GES-14 contains additional hydrogen bonds in the active site formed by oxygen in the side chain of Ser170 with the carboxyl group of Glu166. It is worth noting that GES-11 has a serine at position 170 and, similarly to the previous variants, it shows activity towards carbapenems. Another feature of the amino acid chain of this enzyme is that it has an alanine at position 243, which confers increased resistance to classic β-lactamase inhibitors. Ala243 also makes the enzyme effective against aztreonam, ceftazidime, and cefotaxime (Moubareck et al. 2009).

IR-GES-14 enzyme is a variant of the GES-type β-lactamases, specifically associated with integrons, which enhances GES-14’s ability to spread antibiotic resistance genes. Catalytic serine residue (Ser70) is crucial for the enzyme’s ability to hydrolyze β-lactam rings. It acts as a nucleophile in the hydrolysis reaction. The active site of GES-14, like other GES enzymes, is flexible enough to accommodate a wide range of β-lactam antibiotics (Bonnin et al. 2011).

This enzyme was first identified in S. marcescens in Brazil. The carbapenemase activity of GES-16, like that of other GES variants, is mainly determined by the presence of specific amino acids in its active site. These residues typically include Ser 70, Lys 73, Glu 166 and Gly 170. Based on changes in the amino acid chain, i.e. Gln38Glu and Gly170Ser, GES-16 and GES-5 have been distinguished. In a comparative study regarding the activity of GES-16 against imipenem, ertapenem, and meropenem conducted by Streling et al. (2018), it has been shown that this enzyme has the highest effectiveness against imipenem (compared to other carbap-enems). GES-16, apart from hydrolyzing carbapenems, also inhibits the action of other antibiotics, i.e. penicillin, cephamycin, and cephalosporins, but importantly, it does not hydrolyze aztreonam (Escandón et al. 2017; Streling et al. 2018).

The amino acid sequence in the GES-18 active center is similar to GES-1 and GES-2; the substitution of Gly170Ser and Val80Ile causes a change in the location of the hydrolytic water molecule and the amino acid essential in hydrolysis – glutamic acid (position 166), which may partially explain the differences in enzyme specificity and action. Like GES-5, it has low effectiveness against ceftazidime. Also, it hydrolyzes imipenem and cefotaxime with similar kinetic parameters, while the difference concerns the presence of Val80Ile (in GES-18), but this change does not significantly affect the substrate profile. It is worth noting that GES-18, unlike GES-1, is less susceptible to classic inhibitors, i.e. clavulanic acid and tazobactam (Bebrone et al. 2013).

This type of enzyme was first identified in 2011 in a strain of P. aeruginosa in Mexico (Garza-Ramos et al. 2015). The blaGES-20 gene has two single nucleotide substitutions, translating into amino acid chain changes. Studies have shown that GES-20 resistance often cooccurs with OXA-2 (oxacillinase-2). GES-20-produc-ing isolates studied by Recio et al. (2022) showed the replacement of aspartic acid with serine (position 165). In the place that encodes leucine, a sequence change resulted in the STOP codon (position 237), thus shortening the amino acid chain, translating into resistance to CZA (ceftazidime/avibactam).

In 2024, the World Health Organization (WHO, 2024) published a list of antibiotic-resistant bacteria that pose a considerable threat to public health to indicate the direction for research and development initiatives. WHO has classified bacteria into three risk groups – see Table II.

Bacterial strains exhibiting GES-type antibiotic resistance are treated analogously to other carbapenem-resistant strains. Currently, there are no pharmaceutical agents available that are specifically designed to target this resistance. The following are examples of drugs that can be employed in the treatment of infections caused by carbapenem-resistant bacteria with GES-type resistance, among others.

Given the increasing antibiotic resistance, scientists should develop new drugs that could be used to treat multidrug-resistant strains that threaten humans. A recently developed drug is a siderophore cephalosporin – cefiderocol, which binds to iron, and its action is compared to the mechanism of a “Trojan horse”. Iron is an essential element for the synthesis of bacterial DNA, energy production, and other processes necessary for life. Thus, once this drug binds to iron, it is absorbed by bacteria and then binds to PBP (penicillin-binding protein), its target (Wernicki 2018; European Medicines Agency 2020a).

Other new drugs are eravacycline, a tetracycline, and lefamulin, the first pleuromutilin approved for human use. Lefamulin works by inhibiting the synthesis of bacterial proteins – it blocks the 23S rRNA molecule of the 50S ribosomal subunit (European Medicines Agency 2020b). Eravacycline, a third-generation tetracycline, changes the conformation of ribosomes, preventing protein elongation. This drug has been approved for the treatment of complicated abdominal infections caused by strains producing GES carbapen-emases (and NDM/New Delhi metallo-β-lactamase/, VIM/Verona integron-encoded metallo-β-lactamase/, OXA) (European Medicines Agency 2024a). It is also worth noting that eravacycline is in phase III clinical trials for use in complicated urinary tract infections. In vitro studies conducted by Grossman et al. (2015) showed its promising activity against E. coli. If, in in vivo studies, the effectiveness of this tetracycline on the biofilm formed by the bacteria mentioned above is confirmed. In a second phase III clinical trial, it was discovered that the administration of eravacycline at a dose of 1.5 mg/kg body weight via intravenous infusion every 24 hours, commencing on day 3 with a gradual reduction in dosage to 200 mg administered every 12 hours, was comparable to the use of levofloxacin in the treatment of complicated UTIs (Zhanel et al. 2016).

Carbapenems can also be used in combination with carbapenemase inhibitors against carbapenemase-pro-ducing strains. However, bacteria can cope with this by producing enzymes not susceptible to inhibitors. An example is GES-5 type resistance, characterized by the decreased susceptibility to carbapenemase inhibitors. Therefore, new inhibitors are looked for. For example, the following combinations have recently been developed: relebactam used in combination with imipenem, vaborbactam used in combination with meropenem (Hayden et al. 2020) and ceftazidime with avibactam (Vázquez-Ucha et al. 2020). These three drug combinations have proved safe and effective and should be considered an alternative treatment for infections caused by carbapenem-resistant pathogens (Bouchet et al. 2020).

Relebactam is a DBO (diazobicyclooctane) inhibitor of a second generation (non-β-lactam molecules). It binds to the active site of serine β-lactamases, which effectively inhibits class A (including GES) and C β-lactamases. The drug restores the activity of β-lactams despite bacteria being resistant to these drugs (Bouchet et al. 2020).

Imipenem, in combination with relebactam, has broad activity against many Gram-negative bacteria, including Enterobacterales, P. aeruginosa, and Bacte-roides spp. (belonging to anaerobic bacteria) producing enzymes that inhibit the action of carbapenems. The combination of imipenem and relebactam also shows effectiveness against multidrug-resistant strains resistant to, e.g. fluoroquinolones (Bouchet et al. 2020).

Another important inhibitor is vaborbactam, which is a derivative of boronic acid. Studies have shown that the combination of vaborbactam and meropenem is effective in the treatment of UTIs, nosocomial pneumonia, ventilator-acquired pneumonia, intra-abdominal infections, or bloodstream infections associated with carbapenem-resistant bacteria. Microbiological experiments have shown that adding vaborbactam to mero-penem restores the minimum inhibitory concentration to a level comparable to the wild strain of Enterobacte-rales (Vázquez-Ucha et al. 2020).

When describing new drugs against carbapene-mase-producing strains, the combination of ceftazidime and avibactam should also be mentioned. Avibactam is the first synthetic DBO showing activity against clinically important resistance mechanisms, such as GES, KPC, SHV, CTX-M and OXA-48. The mechanism of action of avibactam, which is based on binding to the active site of the bacterial enzyme, is reversible. After deacylation, an unchanged drug is released, which can inhibit another β-lactamase (including carbapenemase) (Vázquez-Ucha et al. 2020).

GES carbapenemases represent a significant challenge in the treatment of bacterial infections due to their ability to hydrolyze a range of antibiotics, including penicillins, cephalosporins, monobactams and carbapenems (Bonnin et al. 2011). Carbapenems are among the most important antibiotics used to treat multidrug-resistant infections. Developing new drugs against bacterial strains is currently a topic of significant interest. Many drugs are currently under investigation, including durlobactam + sulbactam, taniborbac-tam and cefepime+enmetazobactam. These drugs have demonstrated activity against strains resistant to car-bapenems, including strains with GES-type resistance (Soszyńska-Morys 2023; European Medicines Agency 2024a). Table III shows drugs under investigation for the treatment of infections with carbapenemase-pro-ducing strains of the GES type. The table provides information on the active substances, their class, spectrum of action, testing phases, and additional information on their efficacy and therapeutic areas.

Table IV provides essential data on the antibiotics that can be employed to treat infections caused by carbapenemase-producing strains. The table offers comprehensive information on the active substances, their spectrum of action, and their indications for use. It serves as a valuable reference in daily medical practice to facilitate the selection of an appropriate antimicrobial treatment.