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Molecular diversity of extended-spectrum β-lactamases and carbapenemases, and antimicrobial resistance

Abstract

Along with the recent spread of multidrug-resistant bacteria, outbreaks of extended-spectrum β-lactamase (ESBL) and carbapenemase-producing bacteria present a serious challenge to clinicians. β-lactam antibiotics are the most frequently used antibacterial agents and ESBLs, and carbapenemases confer resistance not only to carbapenem antibiotics but also to penicillin and cephem antibiotics. The mechanism of β-lactam resistance involves an efflux pump, reduced permeability, altered transpeptidases, and inactivation by β-lactamases. Horizontal gene transfer is the most common mechanism associated with the spread of extended-spectrum β-lactam- and carbapenem resistance among pathogenic bacterial species. Along with the increase in antimicrobial resistance, many different types of ESBLs and carbapenemases have emerged with different enzymatic characteristics. For example, carbapenemases are represented across classes A to D of the Ambler classification system. Because bacteria harboring different types of ESBLs and carbapenemases require specific therapeutic strategies, it is essential for clinicians to understand the characteristics of infecting pathogens. In this review, we summarize the current knowledge on carbapenem resistance by ESBLs and carbapenemases, such as class A carbapenemases, class C extended-spectrum AmpC (ESAC), carbapenem-hydrolyzing class D β-lactamases (CHDLs), and class B metallo-β-lactamases, with the aim of aiding critical care clinicians in their therapeutic decision making.

Background

Among the recent spread of multidrug-resistant bacteria, outbreaks of extended-spectrum β-lactam- and carbapenem-resistant bacteria are a serious problem not only making treatment difficult but also worsening the prognosis of infected patients [1]. β-lactam antibiotics are the most frequently used antibacterial agents, and extended-spectrum β-lactams and carbapenems have been developed as specific drugs to treat bacterial species resistant to penicillins and cephems [2]. Bacterial resistance to carbapenems incorporates not only carbapenem antibiotics but also resistance to penicillin and cephem antibiotics. Therefore, resistance to carbapenems presents a significant threat to patients who are immunocompromised and are therefore susceptible to infections caused by multidrug-resistant bacteria all over the world [3]. The mechanism of resistance to extended-spectrum β-lactams and carbapenems involves an efflux pump, reduced permeability, altered transpeptidases, and inactivation by β-lactamases. Horizontal gene transfer is the most common mechanism associated with the spread of antimicrobial resistance across pathogenic bacterial species, such as carbapenemase-producing Enterobacteriaceae (CPE) [4, 5]. In various bacterial species, novel extended-spectrum β-lactamases (ESBLs) or carbapenemases with different structures or characteristic features are reported each year.

Various ESBLs and carbapenemases have been reported in the Enterobacteriaceae including Enterobacter, Klebsiella, Escherichia coli [6, 7], and other opportunistic species such as Serratia, Acinetobacter, and Pseudomonas [8]. In addition, the genetic elements by which drug-resistant genes horizontally move across bacterial species have been studied among these bacterial species. As one typical example, Pseudomonas aeruginosa, one of the major causative agents of infection in immunocompromised individuals, displays resistance to various antibacterial agents. Its resistance mechanism is, in part, derived from the organism’s natural resistance, but is also acquired through horizontal gene transfer and/or mutations within its DNA. P. aeruginosa is naturally resistant to β-lactam antibiotics, such as penicillin and cephem, and aminoglycoside antibiotics. Since the 1990s, P. aeruginosa strains have emerged that have acquired resistance to broad-spectrum penicillins, third-generation cephems, carbapenems, anti-P. aeruginosa aminoglycosides, and new quinolones [9, 10]. Among these multidrug-resistant P. aeruginosa (MDRP), one of the major clinical concerns is the spread of P. aeruginosa strains that harbor a carbapenemase because β-lactam antibiotics including carbapenems are the most frequently used antibacterial agents. An anti-methicillin-resistant Staphylococcus aureus (MRSA) drug, albekacin sulfate, a monobactam aztreonam, and polypeptide colistin appear to be effective against MDRP, but the emergence of resistant strains has also been reported including extensively drug-resistant P. aeruginosa (XDRP) and pandrug-resistant P. aeruginosa (PDRP) [10, 11].

To date, many different types of ESBLs and carbapenemases have emerged with different enzymatic characteristics [1]. Because the specific therapeutic strategy is dependent on the type of ESBL and carbapenemase, it is vital for clinicians to understand the characteristics of ESBLs and carbapenemases [12]; however, in practice, the complex biology associated with ESBLs and carbapenemases presents significant challenges in the effective control of infections. In this review, we summarize antimicrobial resistance by ESBLs and carbapenemases, with the aim of collating the current knowledge in this field to aid therapeutic decision making by critical care clinicians.

β-Lactam antibiotics, penicillin-binding protein (PBP), and β-lactamase

We will begin by reiterating the mechanism of action of β-lactam antibiotics. The main constituent of the cell wall formed in the outer membrane layer of eubacteria is peptidoglycan, which is a macromolecular structure consisting of peptide and sugar (Fig. 1). This peptidoglycan structure confers resistance to osmotic pressure and retains cell morphology and strength. It is also the target of β-lactam drugs. By inhibiting the formation of this structure, β-lactams suppress bacterial cell division (bacteriostatic action) or induce bacterial rupture against osmotic pressure (bactericidal activity). Peptidoglycan possesses a basic structural unit in which two amino sugars of N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM) alternate, and a longitudinal peptide chain linked to NAM forms a pillar. This peptide chain is a pentapeptide consisting of alternating d- and l-forms of l-alanine, γ-d-glutamic acid, l-lysine, d-alanine, and d-alanine residues. At the time of cross-linking, d-alanine at the carboxyl-terminal of pentapeptide is first eliminated by hydrolysis. Next, the fourth d-alanine carboxyl group and the third diaminopimelic acid, which is a ε-carboxyl derivative of lysine of the same columnar structure as the neighboring molecule, are combined with a triglycine peptide structure for horizontal reinforcement of the chain. These reactions are mediated by the bacterial enzyme known as PBP.

Fig. 1
figure1

The antimicrobial action of β-lactamase against the peptidoglycan structure of the bacterial cell membrane. Peptidoglycan possesses a basic structural unit in which two amino sugars of N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM) alternate, and a longitudinal peptide chain linked to NAM forms a pillar. This peptide chain is a pentapeptide consisted of alternating d- and l-forms of l-alanine, γ-d-glutamic acid, l-lysine, d-alanine, and d-alanine that forms a bacterial enzyme known as penicillin-binding protein (PBP). PBP recognizes alanyl-alanine, which is an alanine dimer formed by d-alanine-d-alanine present at the end of the pentapeptide, and exerts the enzymatic action of cross-linking. Since penicillin is structurally similar to alanyl-alanine at the terminal region of the pillar structure, PBP captures penicillin, thereby inhibiting the cross-linking reaction

PBP recognizes alanyl-alanine, which is an alanine dimer formed by d-alanine-d-alanine present at the end of the pentapeptide, and its enzymatic activity mediates cross-linking [13, 14]. A cross-linking reaction is induced by the formation of a covalent bond between the serine residue in the active center of PBP and the carboxyl group produced by hydrolytic cleavage of d-alanine. Thereby, PBP displays adenyl-alanine endopeptidase activity. Since penicillin has a structure similar to that of alanyl-alanine at the terminal part of this pillar structure, PBP cannot distinguish between these structures and binds to both, leading to inhibition of the cross-linking reaction. Seven types of PBP genes exist in the E. coli genome, and eight types of PBP genes exist in the genome of P. aeruginosa reference strain PAO1 (Fig. 2) [15]. Among them, five types of genes (PBP1A, PBP1B, PBP2, PBP3A, and PBP3B) encode high molecular weight PBPs (HMM-PBPs, molecular weight 60,000 to 90,000) that display both transglycosylase and transpeptidase activities and play a role in cell elongation and partition formation. In cell division and morphogenesis, PBP1A and PBP1B are thought to be involved in growth and elongation, PBP2 in formation of a gonococcal form, and PBP3 in partition formation during division.

Fig. 2
figure2

The genes encoding penicillin-binding proteins in P. aeruginosa PAO1. Eight types of PBP genes and three chromosomal β-lactamase genes for PIB-1 (class A), AmpC (class C), and PoxB (class D) exist in the genome of P. aeruginosa reference strain PAO1 [15]

The remaining three types are low molecular mass BPBs (LMM-PBPs; molecular weight of 40,000–50,000) that display d-alanine carboxypeptidase activity and are widely resistant to various β-lactams. The roles of LMM-PBPs are still under research. Both the HMM-PBP transpeptidase activity and the LMM-PBP alanine carboxypeptidase activity utilize a serine residue as an enzymatic activity center, and the β-lactam antibacterial agent exhibits enzyme inhibitory activity by binding to this serine residue, which is a common feature of class A, C, and D β-lactamases (which is discussed again later). As one mechanism of the development of resistant bacteria, the substrate specificity of PBP may be changed to reduce the ability to link to a β-lactam-based antibacterial agent. For example, MRSA produces PBP2′, which possesses a mutation in PBP, that has low affinity for β-lactams. Another example is the mechanism of resistance to the glycopeptide class of antibacterial agents, such as vancomycin. In this mechanism, Enterococci with the vanA gene cluster derived from transposon Tn1546 expresses a group of enzymes that exchange the peptidoglycan peptide longitudinal chain from d-alanine-d-alanine to d-alanine-d-serine and inhibit binding to vancomycin.

Next, we will reiterate the mechanism of action of β-lactamase. β-lactamase inhibits antibacterial activity by dissociating the –CO–NH structure of the β-lactam ring, which is part of the basic structure of β-lactams (Fig. 3). The –CO–NH structure of the β-lactam ring comprises the –CO–NH peptide in the alanyl-alanine dimer from which the peptidoglycan cross-links are formed. Thus, β-lactams mimic the alanyl-alanine of the peptidoglycan pillar structure and this is the binding region. Thus, β-lactamase is a protease (peptidase) that dissociates peptide bonds. PBP is bound by the original substrate alanyl-alanine and penicillin, which works as an inhibitor since it shares a similar structure with alanyl-alanine. These findings indicate that PBP and β-lactamase share similar structures because they exert peptidase activity, implying that β-lactamase may be evolutionarily derived from PBP.

Fig. 3
figure3

The enzymatic action of a β-lactamase to penicillins. The β-lactam ring is the basic structure of β-lactam antibiotics. β-lactamase inhibits the antibacterial action by dissociating the –CO–NH structure of the β-lactam ring. The –CO–NH structure of the β-lactam ring is similar to the –CO–NH peptide in alanyl-alanine, from which the peptidoglycan cross-links are formed. Thus, β-lactams mimic alanyl-alanine of the peptidoglycan pillar structure. Thus, β-lactamase is a protease (peptidase) that dissociates the –CO–NH structure of β-lactams

In fact, the enzyme active site motifs S-X-X-K, S-D-N, and K-S/T-G in the primary sequence of class A β-lactamases, also exist in PBP (Additional file 1: Figure S1). This indicates that the β-lactamases and the peptidoglycan cross-linking enzyme, PBP, co-evolved. At present, clinically used β-lactams can be classified into five basic structures capable of exerting different antimicrobial activities (Fig. 4). Many of them originate from natural antibacterial substances produced by actinomycetes or fungi. Thus, actinomycetes themselves are resistant to their own antimicrobial agents. This indicates that both antibacterial substances and agents that counter such substances co-evolved in actinomycetes.

Fig. 4
figure4

The chemical structure of β-lactam antimicrobials. Clinically used β-lactams can be classified into five basic structures capable of exerting different antimicrobial activities

Classification of β-lactamases and P. aeruginosa drug resistance

To date, β-lactamases have been classified based on the molecular structure classification of Ambler [16] and the functional classification of Bush-Jacobi-Medeiros (Fig. 5) [17, 18]. In the Ambler classification, β-lactamases are grouped into four class A, B, C, and D according to motifs composed of primary sequences constituting the protein molecules. β-lactamases of classes A, C, and D use a serine as an enzyme active center, whereas β-lactamases of class B use the metal zinc. In the functional classification of Bush-Jacobi-Medeiros, β-lactamases are classified into groups 1 to 3 depending on the degradation of β-lactam substrates and the effects of inhibitors. Group 1 comprises cephalosporinases that are classified as class C based on molecular structural classification, and the gene involved was originally chromosomal. Group 2 comprises β-lactamases other than those in group 1 having serine in the active center and includes both classes A and D according to molecular structural classification. Group 3 comprises metallo-β-lactamases (MBLs) and corresponds to class B of the molecular structural classification. Within the genome of P. aeruginosa PAO1, a standard strain of P. aeruginosa and the first strain subjected to whole-genome sequence analysis, the class A PIB-1 (PA5542), class C AmpC (PA 4410), and class D PoxB (OXA-50, PA5541) β-lactamases are encoded (Fig. 2). Thus, P. aeruginosa PAO1 is equipped with three different types of β-lactamases, only lacking a class B β-lactamase [15]. These chromosome-encoded intrinsic β-lactamase genes of P. aeruginosa are partially responsible for its natural resistance to penicillins and cephalosporins.

Fig. 5
figure5

The classification of β-lactamases. Molecular structure classification using the Ambler method [16] and functional classification using the Bush-Jacobi-Medeiros method [17, 18] have both been employed. In the Ambler classification, β-lactamases are grouped into four classes A, B, C, and D by motifs composed of primary sequences constituting the protein molecules. β-lactamases of classes A, C, and D use a serine at the enzyme active center, whereas β-lactamases of class B use metal zinc ions. In functional classification using the Bush-Jacobi-Medeiros method, β-lactamases are classified into groups 1 to 3 depending on the degradation of β-lactam substrates and the effect of the inhibitor

ESBLs and carbapenemases

ESBLs degrade third-generation cephem systems (such as cefotaxime and ceftazidime) [17] and are characterized by whether they are inhibited by β-lactamase inhibitors, such as clavulanic acid, sulbactam, and tazobactam [17]. The appearance of ESBLs in organisms such as Klebsiella and E. coli indicates that β-lactamase-producing genes, such as TEM-type and SHV-type genes, encoded on a plasmid (R plasmid), have broadened the range of target drugs by genetic mutation [12, 19]. Because the resistance genes are derived from the plasmid, they are easily transmitted among different bacterial species belonging to the same genera, such as the Enterobacteriaceae. The repeated mutation of ESBLs has contributed to the emergence of new β-lactamases, namely carbapenemases, which hydrolyze carbapenem antibiotics. In all classes A to D, based on molecular structural classification, β-lactamases that degrade carbapenems have been identified and are discussed below.

Class A penicillinases, ESBLs, and carbapenemases

Class A penicillinases, such as PC1, which belong to the Bush-Jacoby functional subgroup 2a, hydrolyze a relatively limited spectrum of penicillins and are predominantly found in Gram-positive cocci. As class A ESBLs that degrade the early cephalosporins and belong to the Bush-Jacoby functional subgroup 2b, plasmid-mediated TEM-1, TEM-2, and SHV-1 were reported from the 1970s–1980s [20]. Soon after, mutated TEM and SHV ESBLs, such as TEM-3, SHV-2, and CTX-M, which efficiently degrade cefotaxime but remain sensitive to inhibition by clavulanic acid, were reported. These ESBLs belong to the Bush-Jacoby functional subgroup 2be, and since the 1990s, many variants such as BEL-1, BES-1, SFO-1, PER, and VEB were reported as members of this group [21]. TEM-30 and SHV-10, which hydrolyze penicillins and display relative resistance to clavulanic acid, sulbactam, and tazobactam, belong to Bush-Jacoby functional subgroup 2br. TEM-50 is a broad-spectrum lactamase that hydrolyzes extended-spectrum cephalosporins (oxyimino-β-lactams) and monobactams but shows resistance to clavulanic acid, sulbactam, and tazobactam. Enzymes in this group have acquired resistance to clavulanic acid, sulbactam, and tazobactam and belong to Bush-Jacoby functional subgroup 2ber [22, 23]. Class A carbenicillinases and cephalosporinases, which belong to Bush-Jacoby functional group 2, can be further classified into more minor subclasses, such as functional groups 2c, 2ce, and 2e [24]. β-lactamases, such as PSE-1 and CARB-3, belonging to Bush-Jacoby functional subgroup 2c, hydrolyze carbenicillin and are inhibited by clavulanic acid or tazobactam. A subclass of 2c, namely subclass 2ce, comprises one enzyme RTG-4 (CARB-10) that was recently identified as an extended-spectrum carbenicillinase, and hydrolyzes carbenicillin, cefepime, and cefpirome. Subclass 2ce, which includes CepA, hydrolyzes extended-spectrum cephalosporins and is inhibited by clavulanic acid or tazobactam but not aztreonam.

Finally, in the late 1990s, the β-lactamase classified as class A was mutated to become a carbapenemase, which degrades carbapenems [24]. As a feature of class A β-lactamases, the tetrad motif S-X-X-K, the S-D-N triad, and the K-S/T-G triad, which contain the serine of the enzyme active site in their primary sequences, are retained (Additional file 2: Figure S2) [25]. In addition, class A carbapenemases are characterized by susceptibility to inhibition by β-lactamase inhibitors such as clavulanic acid and tazobactam and are classified as Bush-Jacoby functional subgroup 2f [24]. Six types of class A carbapenemases have been reported, these include GES (Guiana extended-spectrum β-lactamase) [26], SME (Serratia marcescens enzyme) [27], SHV (sulfhydryl variable lactamase) [28], KPC (Klebsiella pneumoniae carbapenemase) [29, 30], IMI/NMC-A (imipenemase/non-metallocarbapenemase-A) [31], and SFC (Serratia fonticola carbapenemase) (Fig. 6) [35, 36]. The KPC-type is transmitted via a plasmid and is the most representative example of a class A carbapenemase, it also plays an important role in controlling the characteristics of carbapenem-resistant Enterobacteriaceae bacteria (CRE) [37]. SFC-1 and SME have been reported in Serratia and are closely related to KPC. SME and IMI/NMC-A are chromosomally encoded, and the GES gene blaGES is inserted into the class 1 integron in a plasmid of P. aeruginosa [38].

Fig. 6
figure6

A phylogenetic tree of class A carbapenemases. A phylogenetic tree of six types of class A capbapenemases: GES (Guiana extended-spectrum β-lactamase), SME (Serratia marcescens enzyme), SHV-38 (sulfhydryl variable lactamase), KPC (Klebsiella pneumoniae carbapenemase), IMI/NMC-A (imipenemase/non-metallocarbapenemase-A), and SFC-1 (Serratia fonticola carbapenemase). Based on the primary sequences, the tree was generated using Clustal Omega [32,33,34] of GenomeNet at Kyoto University Bioinformatics Center (https://www.genome.jp/tools-bin/clustalw)

Class C ESAC

The β-lactamase belonging to class C is derived from the ampC gene carried on the genome of many members of the Enterobacteria genus of the Enterobacteriaceae and is functionally a cephalosporinase classified into Bush-Jacoby functional group 1 [39, 40]. It is resistant to clavulanic acid, but sensitive to cephamycins, such as cefoxitin and ceftazidime. Although the expression level of AmpC is usually low, it may be induced by administration of a penicillin system or clavulanic acid and may exhibit carbapenem resistance when it is expressed in large amounts [1]. Some β-lactamases belonging to this group, including CMY, ACT, FOX, and MIR, were encoded on a plasmid [41]. The most significant concern is that variants of AmpC contribute to the reduced sensitivity to carbapenems. These β-lactamases are known as ESAC [42]. In P. aeruginosa, AmpC mutants have been linked to reduced sensitivity to imipenem, ceftazidime, and cefepime. Such mutants, including the plasmid-encoded CMY-10, CMY-19, and CMY-37 mutants, are classified within Bush-Jacoby functional subgroup 1e [43, 44].

The class C β-lactamases share around 40–50% identity at the primary sequence level. The amino-terminal region is the most variable, whereas the carboxyl-terminal region is relatively conserved. They share three characteristic motifs of serine-reactive β-lactamases: S-X-X-K in the enzymatic active site, Y-X-N pointing into the active site, and K-T-G forming the opposite wall of the active site (Additional file 3: Figure S3) [45].

Class D ESBL oxacillinases (OXAs)

β-lactamases of Ambler class D, known as OXA enzymes (Fig. 7), which include OXA-1 and OXA-10, possess an active serine site similar to class A and C β-lactamases. These β-lactamases show cloxacillin- and oxacillin-hydrolyzing activity and are classified into Bush-Jacoby functional group 2d. The overall level of amino acid identity between class D and class A or class C β-lactamases is only about 16%. In β-lactamases such as OXA-11 and OXA-15, the degradation substrate is extended to extended-spectrum cephalosporins (oxyimino-β-lactams), but not carbapenems, and they are classified as Bush-Jacoby functional subgroup 2de [46, 47]. This type of β-lactamases is frequently encoded on plasmids and/or integrons allowing for their wide dissemination [48]. Carbapenem-hydrolyzing class D β-lactamases (CHDLs) are OXA enzymes that hydrolyze carbapenems and belong to Bush-Jacoby functional subgroup 2df. OXA enzymes with carbapenem-hydrolyzing activity have mainly been found on the chromosomes of Acinetobacter baumannii strains [49], but OXA-23 and OXA-48 have been reported on plasmids isolated from enteric bacteria [50, 51]. These OXA-type enzymes are widespread across Acinetobacter, Shewanella, Pseudomonas, and Burkholderi [48, 52]. At the time of writing, 790 OXA-variants could be found in public databases. Similar to class A β-lactamases, class D β-lactamases retain the tetrad motif S-X-X-K (including the serine of the enzyme active site), the Y-G-N/S triad, and the K-T-G triad (Additional file 4: Figure S4) [53].

Fig. 7
figure7

A phylogenetic tree of the class D β-lactamase OXA family (oxiacillinase). A phylogenetic tree of the OXA types of β-lactamases. OXA-10, OXA-11, and OXA-15 are recognized as extended-spectrum cephalosporinases, whereas OXA-23 and OXA-48 are recognized as carbapenem-hydrolyzing class D β-lactamases (CHDLs). Based on the primary sequences, the tree was generated using Clustal Omega [32,33,34] of GenomeNet at Kyoto University Bioinformatics Center (https://www.genome.jp/tools-bin/clustalw)

Class B MBL

Class B β-lactamase is a MBL that possesses the metal Zn2+ at the enzyme active center, compared with a serine residue in the same location for other classes of β-lactamase [54]. Class B β-lactamases hydrolyze carbapenems and belong to Bush-Jacoby functional group 3. P. aeruginosa carrying MBL degrades all β-lactam agents except monobactams. Since the metal is located at the enzymatic active center, its enzymatic activity is suppressed by a chelating agent, such as ethylenediaminetetraacetic acid (EDTA). The MBL gene can reside on an integron, transposon, plasmid, chromosome, or various other genetic molecules.

The components of an integron first reported in 1989 comprised drug-resistant genes of class B MBLs and ESBLs of classes A and D [55,56,57]. Gene cassettes become integrated into the genome via the interaction between two recombination sites (attI and attC), and an integrase catalyzes the genetic recombination (Fig. 8). The promoter, which is located in the integrase gene upstream of the insertion site, controls the transcription of the inserted drug-resistant genes embedded in the cassette structure.

Fig. 8
figure8

The genomic structure of the class 1 integron of P. aeruginosa NCGM2.S1. The intl gene, which encodes integrase Int1, catalyzes recombination between the attl1 site of the integron and the attC site of an antimicrobial gene cassette [56]. In multidrug-resistant P. aeruginosa NCGM2.S1 [58], three antimicrobial gene cassettes (blaIMP-1, aac(6′)-1ae, and aadA1) are integrated into the region between attI and attC. The Int1 integrase gene, the attI integration site, and qacE partially deleted in the sulI gene, which encodes resistance to sulfonamide are present

To date, the carbapenem-resistant metallo-β-lactamases, imipenemase (IMP) [59], Verona integrated-encoded MBL (VIM) [60], Sao Paulo MBL (SPM) [61], Germany imipenemase (GIM) [62], New Delhi MBL (NDM) [63], and Florence imipenemase (FIM) [64] have been reported (Fig. 9, and Additional file 5: Figure S5). Among them, the IMP- and VIM-type MBLs, first discovered in the 1990s, are the major MBLs [60, 65]. New variants of IMP- and VIM-type MBLs are constantly being identified (Additional file 6: Figure S6 and Additional file 7: Figure S7). At the time of writing, 83 IMP- and 66 VIM-variants could be found in public databases. The mutations present in variants affect the spectrum of carbapenem activities, such as activity against imipenem, meropenem, and doripenem [66]. For example, IMP-6, which has only one amino acid substitution (serine to glycine at position 214) from IMP-1 (Additional file 6: Figure S6), enhanced the resistance to meropenem, and meropenem-resistant Enterobacteriaceae, K. pneumoniae, and P. aeruginosa carrying blaIMP-6 have been spreading in Japan since 2001 [67, 68]. Similarly, VIM-4, which has only one amino acid insertion (arginine at position 44) and one amino acid substitution (serine to arginine at position 265) from VIM-1 (Additional file 7: Figure S7), enhanced the resistance to carbapemems, and P. aeruginosa carrying blaVIM-4, which was first reported in 2002, has already been detected worldwide [69, 70]. IMP and VIM are mainly included in the integron structure and are integrated into chromosomal DNA and plasmid DNA in association with the transposon [60, 65]. NDM-1 was detected in Klebsiella and E. coli isolates from patients who returned to Sweden in 2008 following a trip to India, and the blaNDM-1 gene was present on the plasmid [63]. Unlike IMP and VIM, NDM was not found in the integron structure.

Fig. 9
figure9

A phylogenetic tree of the class B metallo-β-lactamase family. MBLs can be classified into three subclasses (B1, B2, B3) based on their amino acid sequences. Subclasses B1 and B3 are characterized by two Zn2+ molecules in the enzyme active center (Zn1, Zn2), implying a broader range of substrates, whereas target substrates of subclass B2, which have a single Zn2+ at the active center, are narrower in range than those of classes 1 and 3 [54]. Based on the primary sequences, the tree was generated using Clustal Omega [32,33,34] of GenomeNet at Kyoto University Bioinformatics Center (https://www.genome.jp/tools-bin/clustalw)

MBLs can be classified into three subclasses (B1, B2, B3) based on their amino acid sequence (Fig. 9) [54, 71]. The level of amino acid identity between groups was as low as 20% or less. Subclass B1 includes IMP, VIM, NDM, and SPM, and subclass B3 includes CAU-1, GOB-1, and FEZ-1, both of which are characterized by the presence of two Zn2+ molecules in the enzyme active center (Zn1, Zn2), degradation of a broader range of substrates, and classification into Bush-Jacoby functional subgroup 3a [54]. The binding site for Zn1 of the B1 enzyme involves three histidines (His116, His118, and His196) [72]. The binding site for Zn2 of the B1 enzyme consists of aspartate, cystine, and histidine (DCH site, Asp-120, Cys-221, His-263) (Additional file 5: Figure S5) [72], whereas the target degradation substrates for CphA, Sfh-I, and ImiS, which are MBLs of subclass B2 having one Zn2+ at the active center, are narrow and these MBLs are classified into Bush-Jacoby functional subgroup 3b [73, 74].

Summary

ESBLs and carbapenemases are represented in all classes, A to D, in the Ambler classification system. Class A carbapenemases, represented by GES and KPC, are encoded on plasmids and are most frequently detected in P. aeruginosa and Klebsiella. As a characteristic feature, they exert an inhibitory effect by β-lactamase inhibitors such as clavulanic acid and tazobactam. The β-lactamases belonging to class C, including ESACs, are encoded by the ampC gene carried on the chromosome of many Enterobacteriaceae, and function as cephalosporinases. In class D, the OXA enzymes, which were originally oxacillinases, have mutated into CHDLs. These enzymes have also been found to be encoded on the chromosome of carbapenem-resistant A. baumannii and on the plasmids of intestinal bacteria such as OXA-23 and OXA-48. Class B β-lactamases are characterized as having a metal Zn2+ in their enzyme activity center. P. aeruginosa carrying an MBL degrades all β-lactam agents, except monobactams. The MBL gene is encoded on an integron, transposon, plasmid, chromosome, or various other genetic molecules. Among them, the IMP- and VIM-type enzymes, first discovered in the 1990s, are the main MBLs that fit into the integron structure. MBLs can be classified into three subclasses (B1, B2, B3) based on their amino acid sequence. Subclass B1 includes IMP, VIM, and NDM. Subclass B3 is characterized by the presence of two zinc molecules (Zn1, Zn2) in the enzyme active center, demonstrating more extensive substrate degradation, whereas subclass B2 has a single Zn2+ in the active center and shows a narrow spectrum of substrates. Figure 10 summarizes carbapenemases of the Ambler classification system [16], including the functional information based on Bush-Jacobi-Medeiros method [17, 18]. Table 1 summarizes the functional classification scheme based on the 1995 proposal by Bush-Jacobi-Medeiros [18] that was updated by Bush-Jacobi [17] in 2010. Together with the Ambler molecular structure classification, antibiotic substrates hydrolyzed by classified β-lactamases and the profiles to β-lactamase inhibitors (clavulanic acid, sulbactam, and tazobactam) are also listed in this table.

Fig. 10
figure10

The classification of carbapenemases. Carbapenemases is represented in all classes, A to D, of the Ambler classification system [16]. Functional classification, using the Bush-Jacobi-Medeiros method [17, 18], indicated that the class A carbapenemases were represented by GES and KPC. The β-lactamases belonging to class C, which function as cephalosporinases, are encoded by the AmpC gene carried on the chromosome of many Enterobacteriaceae. ESAC enzymes are known as ESACs. In class D, OXA enzymes, which were originally oxacillinases, have mutated to become CHDLs. Class B β-lactamases are characterized by possessing a metal Zn2+ as the enzyme activity center. IMP- and VIM-type β-lactamases are the main MBLs that fit into the integron structure. MBLs can be classified into three subclasses (B1, B2, B3) based on their amino acid sequence. Subclasses B1 and B3 are characterized by two zinc molecules (Zn1, Zn2) in the enzyme active center, demonstrating more extensive substrate degradation, while subclass B2 has a single Zn2+ at the active center and displays a narrower spectrum

Table 1 Classification schemes for bacterial β-lactamases by Bush-Jacoby [17, 18]

The following is a list of important issues that clinicians should keep in mind during the daily management of infections:

  1. 1)

    Basic science: PBPs (Fig. 2), which act as adenyl-alanine endopeptidases, enzymatically target an alanyl-alanine of the peptidoglycan pillar structure and catabolize the cross-links between the peptidoglycan layers (Fig. 1). β-lactams (Fig. 4) mimic the alanyl-alanine structure (Fig. 3) and inhibit the enzymatic action of PBPs. β-lactamases share the enzyme active site motifs with the PBP family, indicating that β-lactamases and PBPs have co-evolved (Additional file 1: Figure S1).

  2. 2)

    Classification: β-lactamases have been classified in two ways: one method is based on the molecular structure classification of Ambler, and the other method is based on the functional classification of Bush-Jacobi-Medeiros (Fig. 5). Understanding both structural and functional classifications is important for clinicians in choosing the most appropriate antimicrobial agent (Table 1).

  3. 3)

    ESBLs and carbapenemases: ESBLs and carbapenemases are represented in all classes, A to D, in the Ambler classification system (Figs. 5 and 10). Carbapenemases in all classes have been found on both plasmids and chromosomal genomes. The blaGES gene of class A (Fig. 6 and Additional file 2: Figure S2), blaOXA of class D (Fig. 7 and Additional file 4: Figure S4), and the MBL genes of class B (Fig. 9 and Additional file 5: Figure S5) are frequently encoded in an integron (Fig. 8). The extended-spectrum β-lactamases belonging to class C are encoded by the ampC gene usually carried on the chromosome (Additional file 3: Figure S3).

  4. 4)

    Diversity: The accumulation of small mutations in the primary amino acid sequences of β-lactamases ultimately extends the substrate spectrum. Clinicians can gain insight into how the use of antimicrobial agents may affect the molecular mutation of lactamases by analyzing the alignment of amino acid sequences of specific MBLs, such as IMP (Additional file 6: Figure S6) and VIM (Additional file 7: Figure S7).

  5. 5)

    Diagnostic: Finally, the characterization of antimicrobial resistance is critical for the classification of β-lactam resistance. For more information, please refer to references on the detection of β-lactamase-mediated resistance [75,76,77,78], because this is beyond the scope of this review. In brief, microbiological tests such as the Etest [76], the Clinical and Laboratory Standards Institute (CLSI) method [77] and the Double Disc Synergy Test (DDST) [78] can identify the phenotypes of ESBL, MBL, and ESAC. Novel multiplex polymerase chain reaction (PCR)-based ORF typing (POT) methods that are routinely used in clinical laboratories can identify the prevalent ESBL and carbapenemase genes, such as SHV, GES, TEM, CTX-M, and KPC of class A, NDM, IMP, and VIM of class B, and OXA-48 of class D in A. baumannii [79], P. aeruginosa clones [80], and other pathogenic bacteria. Antimicrobial gene cassettes, which are sometimes integrated into the integron structure via the interaction between two recombination sites (attI and attC), can be identified by PCR amplification with a primer set specific for the consensus sequence (CS) regions (5′-CS and 3′-CS) located upstream and downstream of the insertional site and subsequent DNA sequence analysis [81,82,83,84].

Conclusion

Various ESBLs and carbapenemases belonging to one of four molecular classes have propagated and are being detected among bacteria worldwide, suggesting that careful detection and monitoring are vital when critical care clinicians are treating infections caused by ESBL- and carbapenem-resistant bacteria. For the classification of β-lactamases, the Ambler method of molecular structure classification [16] is simple and effective at organizing the various ESBLs and carbapenemases, but functional classification using the Bush-Jacobi-Medeiros method [17, 18] is also important for clinicians faced with treating patients in a critical condition due to ESBL- and carbapenem-resistant bacterial infections.

Availability of data and materials

All data generated or analyzed during this study are included in this published article and its supplementary information files.

Abbreviations

CHDL:

Carbapenem-hydrolyzing class D β-lactamases

CLSI:

Clinical and Laboratory Standards Institute

CPE:

Carbapenemase-producing Enterobacteriaceae

CS:

Consensus sequence

DDST:

Double disc synergy test

EDTA:

Ethylenediaminetetraacetic acid

ESAC:

Extended-spectrum AmpC

ESBL:

Extended-spectrum β-lactamase

FIM:

Florence imipenemase

GES:

Guiana extended-spectrum β-lactamase

GIM:

Germany imipenemase

HMW-PBP:

High molecular weight penicillin-binding protein

IMI/NMC-A:

Imipenemase/non-metallocarbapenemase-A

IMP:

Imipenemase

KPC:

Klebsiella pneumoniae carbapenemase

LMW-PBP:

High molecular weight penicillin-binding protein

MBL:

Metallo-β-lactamase

MDRP:

Multidrug-resistant Pseudomonas aeruginosa

MRSA:

Methicillin-resistant Staphylococcus aureus

NAG:

N-acetylglucosamine

NAM:

N-acetylmuramic acid

NDM:

New Delhi metallo-β-lactamase

PBP:

Penicillin-binding protein

PCR:

Polymerase chain reaction

PDRP:

Pandrug-resistant Pseudomonas aeruginosa

POT:

PCR-based ORF typing

SFC:

Serratia fonticola carbapenemase

SHV:

Sulfhydryl variable lactamase

SME:

Serratia marcescens enzyme

SPM:

Sao Paulo metallo-β-lactamase

VIM:

Verona integrated-encoded MBL

XDRP:

Extensively drug-resistant Pseudomonas aeruginosa

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Acknowledgements

We thank Kate Fox, DPhil, from Edanz Group (www.edanzediting.com/ac) for editing a draft of this manuscript.

Funding

This work was supported by the Japan Society for the Promotion of Science and a Grant-in-Aid for Scientific Research (KAKENHI) to Teiji Sawa (No. 24390403, No. 15H05008, and No. 18H02905) from The Ministry of Education, Culture, Sports, Science and Technology, Japan.

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TS wrote the manuscript, figure legends, and tables. KK and KM contributed to preparing and revising the manuscript. All authors have read and approved the final manuscript.

Authors’ information

TS is a Professor of the Anesthesiology Department at Kyoto Prefectural University of Medicine, Japan. KK is a Chief Manager in the Division of Critical Care, Kyoto City Hospital, and a Visiting Professor of the Anesthesiology Department at Kyoto Prefectural University of Medicine, Japan. KM is an Associate Professor in the Anesthesiology Department at Kyorin University, Japan.

Correspondence to Teiji Sawa.

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Competing interests

TS has a patent for immunization with PcrV from the Regent of the University of California (Berkeley, CA, USA). All other authors declare that they have no competing interests.

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Sawa, T., Kooguchi, K. & Moriyama, K. Molecular diversity of extended-spectrum β-lactamases and carbapenemases, and antimicrobial resistance. j intensive care 8, 13 (2020). https://doi.org/10.1186/s40560-020-0429-6

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Keywords

  • β-Lactam
  • β-Lactamase
  • Carbapenemase
  • Classification
  • Multidrug resistance