new β-lactamases: a paradigm for the rapid response of bacterial evolution in the clinical setting

10.2217/17460913.1.3.295 © 2006 Future Medicine Ltd ISSN 1746-0913 Future Microbiol. (2006) 1(3), 295–308 295

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New β-lactamases: a paradigm for the rapid response of bacterial evolution in the clinical settingGian Maria Rossolini† & Jean-Denis Docquier†Author for correspondenceUniversity of Siena, Department of Molecular Biology, Section of Microbiology, Policlinico Santa Maria alle Scotte, 53100-Siena, ItalyTel.: +39 057 723 3455;Fax: +39 057 723 3870;[email protected]

Keywords: antimicrobial resistance, β-lactam antibiotics, β-lactamases, β-lactam resistance, carbapenemases, evolution, extended-spectrum β-lactamases, Gram-negative bacteria

Production of β-lactamases is one of the most common mechanisms of bacterial resistance to β-lactam antibiotics. In the clinical setting, the introduction of new classes of β-lactams has invariably been followed by the emergence of new β-lactamases capable of degrading them, as a paradigmatic example of rapid bacterial evolution under a rapidly changing selective environment. The scope of this article is to provide an overview on the recent evolution of β-lactamase-mediated resistance among bacterial pathogens. Focus is on the mechanisms of evolution and dissemination of enzymes of greater clinical impact, including the extended-spectrum β-lactamases, the AmpC-type β-lactamases and the carbapenemases, which are currently responsible for emerging resistance to the most recent and powerful β-lactams (the expanded-spectrum cephalosporins and the carbapenems) among major Gram-negative pathogens such as Enterobacteriaceae, Pseudomonas aeruginosa and Acinetobacter.

Production of β-lactam-degrading enzymes(β-lactamases) is one of the most common mech-anisms by which bacteria defend themselves fromβ-lactam antibiotics. β-lactams are natural prod-ucts, and the production of β-lactamases as aresistance mechanism evolved well before theexploitation of β-lactam derivatives as successfulantibiotics for clinical use. In the antibiotic era,the massive use of β-lactam drugs for chemo-therapy has generated a new and strong selectivepressure for β-lactam-resistant bacteria in the clin-ical setting, and β-lactamases have played a majorrole in the evolution of β-lactam resistance of sev-eral pathogens. Indeed, the relentless introductionof new β-lactams into clinical practice since themid-1940s has invariably been followed by theappearance of new β-lactamases capable ofdegrading them: first the penicillinases and thebroad-spectrum β-lactamases active on both peni-cillins and narrow-spectrum cephalosporins,along with the introduction of penicillins andnarrow-spectrum cephalosporins; then theextended-spectrum β-lactamases (ESBLs) and theAmpC-type β-lactamases, along with the intro-duction of the expanded-spectrum cephalosporinsand monobactams; finally the carbapenemases,along with the introduction of carbapenems [1–3].In fact, the evolution of β-lactamases in the clini-cal setting is one of the most paradigmatic exam-ples of rapid bacterial evolution under a rapidlychanging selective environment.

The scope of this article is to provide an over-view of the recent evolution of β-lactamase-mediated resistance among bacterial pathogens,and some speculation on the potential evolution

of this phenomenon in the near future, focusingon the aspects of greater concern for clinicalpractice. The present paper does not aim to pro-vide a comprehensive and systematic review ofthe various classes of β-lactamases.

Evolution of β-lactam resistance in the antibiotic era: variable contribution of β-lactamases in different pathogensThe antibiotic era has witnessed a rapid evolu-tion of resistance to β-lactam antibiotics amongclinical pathogens, by several mechanisms,including β-lactamase production, penicillinbinding protein (PBP) target modification,decreased outer membrane permeability andactive drug efflux. In this scenario, β-lactamaseshave played a variable role among bacterial patho-gens. In Enterobacteriaceae, β-lactamases havealways played a predominant role in acquiredβ-lactam resistance, covering all major classes ofpotentially useful β-lactams [4,5]. In Pseudomonasaeruginosa and Acinetobacter spp., β-lactamaseshave also played a pivotal role in acquired resist-ance to all major classes of β-lactams; however,the contribution of impermeability, active efflux(and, possibly, PBP target modification, inAcinetobacter) was also remarkably important inthese species [4,6]. On the other hand, inStaphylococcus aureus and Hemophilus influenzae,β-lactamase production has been responsible forresistance only to penicillins and narrow-spec-trum cephalosporins (in the latter species), whiletarget modification has also evolved as a majormechanism of resistance to other classes ofβ-lactams [7,8]. Finally, in Enterococcus faecalis

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296 Future Microbiol. (2006) 1(3)

and in Streptococcus pneumoniae, β-lactamaseproduction has either played a very marginal roleor none at all, respectively, as a mechanism ofacquired β-lactam resistance [8,9].

Currently, the contribution of greater clini-cal relevance is provided by β-lactamases capa-ble of hydrolyzing the most recent andpowerful β-lactams (i.e., the expanded-spec-trum cephalosporins and the carbapenems)among major Gram-negative pathogens such asEnterobacteriaceae, P. aeruginosa and Acineto-bacter spp. Considering that, taken together,these pathogens account for approximately47% of isolates from in-patients and 62% fromout-patients [10], and that the expanded-spectrum cephalosporins and carbapenems areamongst the most important therapeutic optionsfor these infections, the potential for clinicalimpact of β-lactamase-mediated resistance isreadily evident.

Clinically relevant β-lactamases emerging at the beginning of the new centuryAn operative classification of the β-lactamasesthat are currently spreading in Gram-negativepathogens and represent a matter of clinical con-cern is reported in Table 1. The nomenclature ofβ-lactamases is not straightforward, and somedefinitions (such as that of ESBLs) have beenused with different meanings by differentauthors. In this article the following terminologyis used:

• ESBLs refers to enzymes belonging to molecularclasses A or D that exhibit an extended substratespecificity including the expanded-spectrumcephalosporins and monobactams;

• AmpC-type β-lactamases refers to enzymesbelonging to molecular class C that arecapable of degrading expanded-spectrumcephalosporins;

• Carbapenemases refers to those β-lactamasesthat are capable of efficiently degrading carba-penems (usually in addition to other β-lactamsubstrates) regardless of their molecular class.

Each molecular class of β-lactamases has con-tributed enzymes capable of degrading theexpanded-spectrum cephalosporins, while threeof the four classes (A, D and B) have contributedenzymes with carbapenemase activity, showingthat all classes of β-lactamases have played a rolein the evolution of resistance to the most recentand powerful β-lactams.

The following sections will consider themechanisms of evolution and dissemination ofrepresentative members of these groups ofenzymes as paradigms of the recent evolution ofβ-lactamase-mediated resistance in majorGram-negative pathogens.

Extended-spectrum β-lactamases in EnterobacteriaceaeThe ESBLs were first identified in Enterobacte-riaceae in the mid-1980s as enzymes encoded bytransferable plasmids and capable of conferringresistance not only to penicillins and narrow-spectrum cephalosporins (as was the case for thealready widespread broad-spectrum β-lactamasessuch as the TEM-1, TEM-2 and SHV-1enzymes) but also to the expanded-spectrumcephalosporins and monobactams [11,12]. Theselection for these enzymes was apparently conse-quent to the introduction of oxyimino-cephalo-sporins in clinical practice since the early 1980s,

Table 1. Clinically relevant β-lactamases emerging in Gram-negative pathogens.

Enzyme group Molecular class Distribution Epidemiological impact

Extended-spectrum β-lactamases (ESBLs)

A Enterobacteriaceae High

GNNFs* Moderate‡

D GNNFs Very low ‡

AmpC-type β-lactamases C Enterobacteriaceae Moderate

Carbapenemases A Enterobacteriaceae Low‡

B Enterobacteriaceae Low

GNNFs Moderate

D Enterobacteriaceae Very low‡

Acinetobacter spp. Moderate

*GNNF: Nonfastidious Gram-negative nonfermenters (e.g., Pseudomonas aeruginosa, Acinetobacter spp.).‡Distribution is restricted to some geographical areas.See text for a detailed definition of enzyme groups.

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and was gradually recognized as a matter of majorconcern for antimicrobial chemotherapy, sinceESBL production significantly narrows the reper-toire of therapeutic options. In fact, amongβ-lactams, only carbapenems retain reliable activ-ity against ESBL producers, while these strainsare often co-resistant to non-β-lactam agents suchas aminoglycosides and fluoroquinolones [13].

According to data from recent surveillancestudies, ESBL production in Enterobacteriaceae isnow a problem of global dimensions, especiallyamong Klebsiella pneumoniae and Escherichia coli,with remarkably high rates among clinical isolatesfrom some areas (e.g., Europe, South Americaand Asia) [14–17]. In fact, among the β-lactamasesemerging in Enterobacteriaceae, the ESBLs aredefinitely those of greatest epidemiological andclinical impact.

Characterization of the first ESBLs detected inEnterobacteriaceae revealed that they were mutantsof the TEM- and SHV-type broad-spectrum

β-lactamases, the most prevalent types of plas-mid-encoded broad-spectrum β-lactamasesamong Enterobacteriaceae worldwide, which werealso the major cause of acquired resistance topenicillins and narrow-spectrum cephalosporinsin species that are naturally susceptible to thosecompounds (e.g., E. coli, Proteus mirabilis andSalmonella enterica) [18].

In TEM-type ESBL mutants, extension ofthe substrate spectrum is achieved by single ora few amino acid substitutions that have theeffect of broadening the active site of thenative enzyme [2,19], which becomes moreaccessible to the expanded-spectrum cepha-losporin molecules carrying bulky R1 oxy-imino side chains (Figure 1). Mutationsentailing this effect are largely clustered inthree structural domains:

• The loop containing residue Glu-104, whichdelimitates the left side of the entrance of thebinding site;

• The ω-loop, which forms the bottom of thebinding site;

• The terminal part of the β3 strand, which formsthe right edge of the binding site (Figure 2).

In each of these elements, mutations of keyresidues (Figure 2) can improve the catalytic effi-ciency of the enzyme toward expanded-spectrumcephalosporins, while combined mutations canfurther improve the catalytic efficiency of theenzyme toward the same substrates (Table 2),resulting in successful TEM-type ESBLs such asTEM-26 [13,20]. Mutations expanding the sub-strate specificity, however, are often not neutral,but associated with a reduction of the catalyticefficiency towards penicillins and of protein sta-bility (Table 2) [21,22]. While the former issue doesnot significantly affect the ability of the enzymeto confer clinical resistance to penicillins,decreased stability can eventually impact on theenzyme efficiency in conferring resistance [22].The latter phenomenon can be circumvented bycompensatory mutations such as the Met-182-Thr substitution, which does not contributeper se to expand the substrate specificity but actsas a global suppressor of the stability defects(Table 2) [22,23]. In fact, TEM-type variants withthe Met-182-Thr suppressor, such as TEM-52/92[24,25] or TEM-72 [26], are powerful and successfulESBLs that have become highly prevalent in somesettings [27,28].

With the SHV-type ESBL variants the situa-tion is similar: even in this case the substratespectrum of the native enzyme (such as SHV-1)

Figure 1. Three cephalosporin antibiotics.

The nature of the R1 and R2 substituents in narrow-spectrum cephalosporins (cephalothin), and expanded-spectrum cephalosporins containing a bulky oxyimino group at position R1 (cefotaxime and ceftazidime).

S

N

SNH

O

COO-

O

O

R1

R2Cephalothin

S

N

N

SNH

O

COO-

O

O

NO

H2N

R2

R1

Cefotaxime

S

N

N

SNH

O

COO-

N+

O

NO

H2N

COO-R2

R1

Ceftazidime



can be extended by single or multiple amino acidsubstitutions that affect the conformation of thebinding site and render it more accessible tobulkier expanded-spectrum cephalosporin mole-cules [20,29,30]. However, despite the fact thatSHV-type β-lactamases are highly similar toTEM-type enzymes in structure and can evolveESBL activity by a similar strategy, modulationof substrate specificity by mutation may followdifferent pathways in the two enzymes. Forinstance, in the ω-loop, the preferred hotspot formutations extending the substrate specificity isat position 164 in TEM-type enzymes and atposition 179 in SHV-type enzymes [31]. Thisreflects the importance of the protein back-ground in which analogous mutations couldoccur, in terms of eventual selective advantageconferred by the mutant enzyme to the bacterialhost [31].

Modulation of substrate specificity of pre-existing TEM- and SHV-type enzymes by pointmutations has been a major mechanism of ESBLevolution in Enterobacteriaceae. However, sincethe early 1990s, an additional mechanism ofESBL evolution was consistently adopted, basedon the capture of new ESBL-encoding genesfrom the heterologous metagenome. A numberof such ESBLs have been described, includingmembers of the CTX-M, PER, VEB, GES, TLA,SFO and BES lineages [13]. While most of theseenzymes have only been reported sporadically oras causing outbreaks with limited geographicaldistribution, the CTX-M-type ESBLs have rap-idly spread worldwide and, in several epidemio-logical settings, are now among the mostcommon ESBLs in E. coli and/or K. pneumoniae,often outnumbering the TEM- and SHV-typeESBLs [13,32–35].

Figure 2. Ribbon representation of the 3D structure of the TEM-1 β-lactamase (PDB entry, 1BTL).

The enzyme molecule is made by an α-helical domain (on the left) and a mixed α/β domain (on the right). The binding site for β-lactam molecules is located in a cleft between the two domains, and is delimited by three structural elements: the loop containing position 104 (pink), on the left side, the Ω-loop (green), at the bottom, and the β3 strand (red), on the right side. The principal residues where mutations expanding the substrate specificity occur are shown as red dots, and the most common substitutions observed in extended-spectrum β-lactamases variants are also indicated (amino acids are indicated using the one-letter code). These amino acid substitutions result in broadening of the binding site, which can more easily recognize the bulkier expanded-spectrum cephalosporin molecules. The active site serine residue (S*70), located at the extremity of the α2 helix (yellow) is also shown (blue dot).The diagram was constructed using the MOLMOL program [78].

loop

E104

S*70S*70

K

R164

SHC

G238

SD

G238

E240

KRV

E240

2

3



The original source of these ESBL genesremains unknown, except for those encodingCTX-M-type enzymes. The latter are derived fromchromosomal class A β-lactamase genes from Kluy-vera spp. [36–39], an enterobacterial genus of overallvery low pathogenic potential for humans [40]. Inmost cases, blaCTX-M genes are found associatedwith a particular type of insertion sequences, suchas ISEcp1 and ISCR1, that are capable of mobiliz-ing flanking DNA regions by a peculiar transposi-tion mechanism [41,42]. This points to a major roleof these elements in capturing blaCTX-M genes onboard of conjugative plasmids and, possibly, also intheir further spreading. Recently, a blaCTX-M genewas also found associated to phage-relatedsequences within a different genetic context [43],suggesting the involvement of additional elementsin the mobilization of these ESBL genes.

The reason(s) for the rapid and massive dis-semination of CTX-M-type ESBLs in Entero-bacteriaceae, observed during the past decade[13,32–35], remains unknown. Carriage on plas-mids that are particularly efficient at spreadinghorizontally by conjugation, and/or a lower fit-ness cost imposed by these enzymes and the cog-nate genetic elements to the bacterial host, ascompared with other ESBLs, could be hypo-thesized to be among the possible causes for theremarkable success of CTX-M-type enzymesversus other types of ESBLs.

Unlike the precursors of the TEM- and SHV-type ESBLs, the CTX-M-type enzymes are natu-ral ESBLs, although with a strong preference for

some expanded-spectrum cephalosporins (cefo-taxime and ceftriaxone) [32]. The molecular foldof these enzymes overall resembles that of otherclass A β-lactamases (Figure 3), but unlike inTEM-1 and SHV-1, the geometry of the activesite of CTX-M-type enzymes enables an efficientrecognition of cefotaxime [44,45], which is a sub-strate as good as penicillins and narrow-spectrumcephalosporins (Table 3). On the other hand, theactive site of the CTX-M-type enzymes is notreadily accessible to the bulkier ceftazidimemolecule (Figure 1), which remains a very poorsubstrate (Table 3), thus explaining the peculiarsubstrate specificity of these ESBLs.

Modulation of enzyme activity by point muta-tions, as observed with the TEM- and SHV-typeenzymes, is also possible with the CTX-M-typeenzymes. A similar phenomenon is exemplifiedby the emergence of CTX-M-type mutants withincreased ceftazidimase activity, which have likelybeen selected by the massive use of ceftazidime inclinical practice. Mutations leading to increasedceftazidimase activity occur either in the terminalpart of the B3 β-strand or in the ω-loop (Figure 3).The Asp-240-Gly substitution would be responsi-ble for an increased flexibility of the B3 β-strandrendering the active site more accessible to thebulkier ceftazidime molecule [45], while the Pro-167-Ser substitution might cause a change in theinteraction modes of β-lactams with the bindingsite [46]. In both cases, the increase in catalytic effi-ciency is modest overall (Table 3), but it is suffi-cient to significantly increase ceftazidime

Table 2. Kinetic parameters for the hydrolysis of cefotaxime and ceftazidime by single and complex TEM extended-spectrum β-lactamases mutants, and their relative stability.

Enzyme Mutation(s) kcat/Km (M-1.s-1)* Relative stability§

Cefotaxime Ceftazidime

TEM-1 2.1 × 103 3.2 × 101

TEM-17 Glu-104-Lys 9.4 × 103 (5) 5.4 × 102 (17) -0.22

TEM-12 Arg-164-Ser 8.8 × 103 (5) 5.3 × 103 (160) -0.73

TEM-19 Gly-238-Ser 1.8 × 105 (86) 6.1 × 102 (19) -1.94

TEM-26 Glu-104-Lys; Arg-164-Ser 6.0 × 104 (30) 1.6 × 105 (5100) -0.26

TEM-15 Glu-104-Lys; Gly-238-Ser 1.7 × 106 (810) 9.1 × 103 (280) -2.24

TEM-52 Glu-104-Lys; Met-182-Thr; Gly-238-Ser 1.0 × 106 (500) 4.3 × 104 (1300) 1.76

‡ Met-182-Thr 9.4 × 103 (5) 5.0 × 101 (1.6) 2.67

*The variation of catalytic efficiencies of mutants, in comparison with TEM-1, is expressed as and is given between brackets.

‡Laboratory mutant. Now appearing in the Lahey website [101] as TEM-135.§The relative stability is given as the ration of the variation of free energy of unfolding relative to TEM-1 (ΔΔGu, kcal.mol-1), as described in [22].

Data are from [22].

kcat Km⁄( )TEM X–

kcat Km⁄( )TEM 1–

-------------------------------------------



minimum imhibitory concentrations (MICs) forthe isolates producing the mutant enzymes com-pared with those producing the parent enzymes(Table 4). However, while the Asp-240-Gly muta-tion does not significantly affect the activity oncefotaxime, the Pro-167-Ser mutation is associ-ated with a significant decrease in the catalyticefficiency for other substrates (Table 3), which isalso reflected in MIC values (Table 4). The ω-loopof the CTX-M-type enzymes, therefore, wouldappear a less tolerant target for substitutionsmodulating the substrate spectrum than that ofTEM- or SHV-type β-lactamases, and this wouldprobably account for the fact that emergence ofthis type of mutant has been less common overall.

AmpC-type β-lactamases in EnterobacteriaceaeIn Enterobacteriaceae, acquired resistance toexpanded-spectrum cephalosporins can also bemediated by production of AmpC-type

β-lactamases. In general, these enzymes can con-fer resistance to the third-generation cephalo-sporins (e.g., cefotaxime, ceftriaxone andceftazidime) while demonstrating a relatively pooractivity toward the zwitterionic fourth-generationcephalosporins (e.g., cefepime) [18,47,48].

A number of clinically relevant enterobacterialspecies (e.g., Enterobacter spp., Citrobacter freundii,Serratia marcescens, Morganella morganii and Prov-idencia stuartii) produce inducible AmpC-typeβ-lactamases, encoded by resident chromosomalgenes, that are responsible for intrinsic resistanceto penicillins and narrow-spectrum cephalosporins(that are both substrates and inducers of theseenzymes) and can cause acquired resistance tothird-generation cephalosporins (which are sub-strates but not inducers) if enzyme production isderepressed by chromosomal mutations [18].

In addition, there are at least six different line-ages of AmpC-type β-lactamase genes that haveescaped to plasmids that are spreading among

Figure 3. Ribbon representation of the 3D structure of the CTX-M-9 β-lactamase (PDB entry, 1YLJ).

The molecular fold and the structural elements defining the β-lactam binding site are similar overall to those of TEM-type enzymes (see description in legend to Figure 2). In CTX-M-type enzymes, the positions of amino acid substitutions observed in variants with increased ceftazidimase activity are located either in the Ω-loop (green, at the bottom of the binding site) or at the end of the β3 strand (red, on the right side of the binding site), and are indicated as red dots (amino acids are indicated using the one-letter code). The active site serine residue (S*70), located at the extremity of the α2 helix (yellow) is also shown (blue dot). The diagram was constructed as described in the legend to Figure 2.

S*70S*70

P167

S

P167

D240

G

D240loop

2

3



Enterobacteriaceae (Table 5). These acquiredAmpC-type enzymes can confer a β-lactamresistance phenotype similar to that observed inderepressed mutants of species carrying residentampC genes, and also in species that do not typi-cally express this type of β-lactamase [48]. Theseplasmid-mediated AmpC-type β-lactamases havemostly been detected in E. coli, K. pneumoniae,S. enterica and P. mirabilis, although some havealso been reported in other enterobacterial spe-cies, and most of them often exhibit a wide-spread distribution (Table 5). The actualprevalence of these enzymes could possibly beunderestimated, since their presence wouldprobably not be spotted in species that alreadyexpress a chromosomal AmpC-type enzyme.

In most cases, the original source of plasmid-mediated AmpC-type β-lactamase genes isknown, being represented by chromosomalampC genes of a member of the family Entero-bacteriaceae or of genus Aeromonas (Table 5).Multiple elements have apparently beeninvolved in the mobilization of these genes andcan contribute to their further dissemination,including composite transposons generated byIS26 (e.g., in the case of blaCMY-13 [49]) andinsertion sequences capable of mobilization offlanking DNA sequences by one-ended transpo-sition mechanisms such as ISEcp1 (e.g., in thecase of blaCMY-2 [50]) or ISCR1 (e.g., in the caseof some blaCMY/MOX allelic variants and ofblaDHA-1 [42,51,52]).

Table 3. Kinetic parameters for the hydrolysis of β-lactam substrates of CTX-M-type enzymes representative of the various subgroups, and for mutants carrying substitutions affecting activity against ceftazidime.

Enzyme (subgroup) Mutation kcat/Km (M-1.s-1)

Penicillin G Cephalothin Cefotaxime Ceftazidime

CTX-M-3 (CTX-M-1) 1.1 × 108 3.0 × 107 3.5 × 106 ND

CTX-M-15 CTX-M-3 Asp-240-Gly 4.0 × 106 5.0 × 105 3.0 × 106 1.0 × 103

TOHO-1 (CTX-M-2) 3.0 × 106 1.2 × 107 2.1 × 106 1.3 × 103

CTX-M-8 (CTX-M-8) 1.4 × 107 1.9 × 107 9.7 × 105 4.0 × 103

CTX-M-9 (CTX-M-9) 1.2 × 107 2.0 × 107 3.7 × 106 3.3 × 103

CTX-M-16 CTX-M-9 Asp-240-Gly 1.1 × 107 3.4 × 107 9.3 × 106 4.3 × 104

CTX-M-18 (CTX-M-9) 1.0 × 106 3.0 × 104* 3.7 × 105 ND

CTX-M-19 CTX-M-18 Pro-167-Ser 3.0 × 105 2.5 × 105* 5.5 × 104 1.0 × 102

*Data for cephaloridine.ND: Activity not detectable.Data are from [32,44,45,79].

Table 4. Susceptibility of Escherichia coli strains producing various CTX-M-type β-lactamases.

β-lactamase produced MICs (µg/ml)

Cefotaxime Ceftazidime Cefepime

None 0.03 0.12 0.06

CTX-M-1 >256 6 48

CTX-M-32 (CTX-M-1 Asp-240-Gly) >256 >256 64

CTX-M-3 512 32 128

CTX-M-15 (CTX-M-3 Asp-240-Gly) 512 256 64

CTX-M-9 16 1 NA

CTX-M-16 (CTX-M-9 Asp-240-Gly) 16 8 NA

CTX-M-18 64 2 16

CTX-M-19 (CTX-M-18 Pro-167-Ser) 4 128 4

Note: This table illustrates the effect of mutations at positions 240 and 167 on expanded-spectrum cephalosporin MICs (each couple of parent and mutant enzymes were produced in an isogenic background).MIC: Minimum inhibitory concentration; NA: Data not avaliable.Data are from [32,80,81].



Modulation of enzyme activity by mutations,as observed with the TEM-, SHV- and CTX-M-type enzymes, is also possible with AmpC-typeβ-lactamases. A similar phenomenon is exempli-fied by the emergence of mutants with increasedactivity against fourth-generation cepha-losporins, which have likely been selected by theuse of these agents in clinical practice. A typicalexample is that of CMY-19, a variant of theCMY-9 enzyme with an Ile-292-Sersubstitution [52]. Compared with CMY-9,CMY-19 exhibits a significantly increased cata-lytic efficiency against cefepime and can confer ahigher resistance to this drug to the bacterial host(Table 6). The point mutation also affects theenzyme activity against other β-lactam substratesincluding third-generation cephalosporins(Table 6). Other examples of mutational modula-tion of the substrate profile of AmpC-typeβ-lactamases, with increased activity againstfourth-generation cephalosporins, have beenreported with chromosomally encoded enzymesfrom S. marcescens [53], E. coli [54], Enterobacter

aerogenes [55], and Enterobacter cloacae [56]. In allcases, mutations (small deletions or substitu-tions) are clustered in the region of the R2-loop,which defines one edge of the active site ofAmpC-type enzymes [57].

Acquired carbapenemases in Enterobacteriaceae and nonfastidious Gram-negative nonfermentersAt the time of their introduction in clinicalpractice in the mid-1980s, carbapenems wereknown for being exceptionally stable to β-lacta-mases produced by major clinical pathogens,including Enterobacteriaceae and P. aeruginosa, afeature that contributed to their very broad anti-microbial spectrum [58]. In those pathogens,acquired carbapenem resistance mediated byproduction of carbapenemases emerged only inthe early 1990s and remained relatively uncom-mon, especially in Enterobacteriaceae [59,60]. InP. aeruginosa, where acquired carbapenem resist-ance became an issue shortly after carbapenemintroduction, the first documented mechanisms

Table 5. Major plasmid-encoded AmpC-type β-lactamases currently spreading in the clinical setting.

Enzyme lineage

Geographical distribution

Hosts Gene source Ref.

CMY/LAT AF/ME, AU, EU, FE, NA Escherichia coli Citrobacter freundii [48,82–84]

Klebsiella spp.

Proteus mirabilis

Salmonella enterica

Enterobacter aerogenes

DHA AF/ME, EU, FE, NA Escherichia coli Morganella morganii [48,82,85]

Klebsiella spp.

Proteus mirabilis

Salmonella enterica


ACC AF/ME, EU, NA Escherichia coli Hafnia alvei [48,86]

Klebsiella pneumoniae

Proteus mirabilis

Salmonella enterica

CMY/MOX EU, FE Escherichia coli Aeromonas spp.? [48,52,87]

Klebsiella spp.


FOX EU, NA, SA Escherichia coli Aeromonas caviae [48,88,89]

Klebsiella spp.

Enterobacter cloacae

ACT/MIR FE, NA Escherichia coli Enterobacter spp. [48,82,85]

Klebsiella pneumoniae

AF/ME: North Africa and/or Middle East; AU: Australia; EU: Europe; FE: Far East; NA: North America; SA: South America.



of acquired carbapenem resistance (and still themost common ones) were represented byreduced outer membrane permeability and/oractive efflux of the drug from the periplasmicspace following chromosomal mutations [61].This belated emergence and relatively slowspread of carbapenemases in the clinical setting,compared with that observed with other types ofβ-lactamases (e.g., the ESBLs) probably reflect,at least in part, a difficulty in evolving efficientcarbapenemase activity by enzymes that wereprevalent in the clinical setting at the time of car-bapenem introduction in medical practice. Infact, although some variants of class A β-lacta-mases of the SHV- and GES-types (e.g., SHV-38and GES-2) have evolved some weak carbapene-mase activity (which can contribute to resistancewhen production of the enzyme is associatedwith a permeability defect [62,63]), the evolutiontoward efficient carbapenemase activity hasnever been demonstrated for the highly preva-lent class A enzymes of the TEM and SHV typesor for the AmpC-type β-lactamases.

Under these conditions, capturing new car-bapenemase-encoding genes from the environ-mental metagenome remained the onlyalternative to evolve carbapenemase production.This phenomenon apparently evolved at a rela-tively slow pace, possibly due to a smaller dimen-sion of the natural carbapenemase gene pooland/or to major constraints encountered inmobilizing these genes from remote species andhaving them transferred, stably maintained andexpressed in Enterobacteriaceae, P. aeruginosa andother nonfastidious Gram-negative nonferment-ers of clinical significance. Nevertheless, anumber of acquired carbapenemases belongingin three different molecular classes (A, D and B)have been captured in the clinical setting and

are currently spreading among Enterobacte-riaceae, P. aeruginosa and other nonfastidiousGram-negative nonfermenters (Table 1).

The acquired class A carbapenemases includeat least three different lineages of enzymes (SME,NMC/IMI and KPC), which have been detectedin clinical and environmental isolates of Entero-bacteriaceae [60,64]. The SME- and NMC/IMI-type enzymes have mostly been detected in spo-radic isolates [60,65], and their clinical impactappears to be low overall. On the other hand, theKPC-type enzymes (mostly detected inK. pneumoniae, but also in other species) haverecently been involved in nosocomial outbreaksin US hospitals [66,67], demonstrating the poten-tial for a greater clinical impact. From the func-tional standpoint, the class A carbapenemasescan efficiently degrade and confer resistance tocarbapenems and penicillins, while activityagainst cephalosporins is more variable [60].Some of these enzymes, such as KPC-2, exhibita very broad substrate specificity and can conferresistance to carbapenems, penicillins,expanded-spectrum cephalosporins and mono-bactams [68]. The original sources of genesencoding acquired class A carbapenemasesremain to be clarified.

The acquired class D carbapenemases includeat least four different lineages of enzymes(OXA-23, OXA-40, OXA-48 and OXA-58), andhave mostly (although not exclusively) beendetected in Acinetobacter [69,70]. Some of theseenzymes have only been reported sporadically,while others (such as OXA-23 and OXA-58) havea broader epidemiological impact [70]. Comparedwith other carbapenemases, class D enzymes areoverall less efficient in degrading carbapenems,usually being more active against imipenemthan meropenem [70], and their ability to confer

Table 6. Kinetic parameters of CMY-9 β-lactamase and its Ile-292-Ser mutant (CMY-19).

Enzyme kcat/Km (M-1.s-1)

Cefotaxime Ceftazidime Cefepime

CMY-9 9.6 × 105 3.2 × 103 ND

CMY-19 (CMY-9 Ile-292–Ser) 1.1 × 104 2.3 × 104 2.9 × 103

β-lactamase produced Escherichia coli MICs (µg/ml)

None <0.06 <0.06 <0.06

CMY-9 >128 64 0.12

CMY-19 128 >128 4

Note: This table illustrates increased activity on cefepime, and impact on CMY-type enzymes on in vitro antimicrobial susceptibility.MIC: Minimum inhibitory concentration; ND: Activity not detectable.Data are from reference [52].



carbapenem resistance would largely be dependenton the presence of a strong permeability barrier forcarbapenems. This would explain the ability ofOXA-type carbapenemases to confer carbapenemresistance in Acinetobacter but usually not in otherspecies [70]. The original sources of genes encodingacquired class D carbapenemases remain unknown.However, a resident gene encoding a protein verysimilar to OXA-48 has been detected in Shewanellaoneidensis, suggesting that some closely related spe-cies could be the source for that type of class Dcarbapenemases [71]. In many Acinetobacter strains,a chromosomal class D carbapenemase gene(blaOXA-51 lineage) is also present [72,73]. This geneis normally expressed at low levels and does notconfer carbapenem resistance. However, acquiredcarbapenem resistance can apparently be mediatedby over-expression of this gene [74].

The acquired class B carbapenemases are met-allo-β-lactamases (MBLs), which belong in at leastfive different lineages (IMP, VIM, SPM, GIM andSIM) [75,76]. The IMP- and VIM-type enzymes, forwhich multiple allelic variants are known [75], havebeen detected worldwide both in Enterobacte-riaceae and in nonfastidious Gram-negative non-fermenters, not only sporadically but alsoassociated with small and even large nosocomialoutbreaks [77]. The three other MBL types have onlybeen found in P. aeruginosa (SPM and GIM) [75] orin Acinetobacter (SIM) [76], and their epidemiologi-cal impact appears more limited to date, althoughSPM is achieving a significant spread in Brazil [75].The powerful carbapenemase activity of acquiredMBLs (kcat/KM ratios for carbapenems are in therange of 105–106 M-1.s-1), along with theirexceedingly broad spectrum (which includes vir-tually all β-lactams except monobactams) andunsusceptibility to therapeutic serine-β-lactamaseinhibitors [75,76], render these enzymes particularlyworrisome from the clinical standpoint. Even inthis case, the original sources of these genes remainunknown, likely being represented by species fromthe environmental microbiota. The fact thatacquired MBL genes were initially found in P. aer-uginosa and in other Gram-negative nonferment-ers, and are overall more common in these speciesthan in Enterobacteriaceae, would suggest thatGram-negative nonfermenters are the primaryentry port for these genes in the clinical setting. Asprimarily environmental species, the Gram-nega-tive nonfermenters could share the same ecologicalniches with the donors of the genes, and as oppor-tunistic pathogens they could shuttle to the hospi-tal microbiota the genes acquired from theenvironmental reservoirs.

The mechanisms of capture and spread ofacquired carbapenemase genes are only partiallyunderstood. Class D carbapenemase genes areoften associated with insertion sequences thatcould be involved in their mobility and also in theirexpression [70,74]. On the other hand, mostacquired MBL genes (namely those of the IMP,VIM, GIM and SIM types) are carried on mobilegene cassettes inserted into integrons [75,76], reveal-ing that the integron system has played a major rolein their dissemination.

ConclusionsProduction of β-lactamases is a major mechanism ofresistance to the most recent and powerful β-lactams(expanded-spectrum cephalosporins and carbapen-ems) among Enterobacteriaceae, P. aeruginosa andAcinetobacter, with a remarkable clinical impact.

In these species, the recent evolution of β-lacta-mase-mediated resistance has followed multiplestrategies including:

• Modulation of the spectrum of activity of existingenzymes by mutations;

• Mutational or IS-mediated over-expression ofresident enzymes;

• Capture of new β-lactamase genes from the nat-ural β-lactamase gene pool via mechanisms ofhorizontal gene transfer that can be mediated byseveral different types of mobile genetic elementsand recombination systems.

Such a complex evolutionary pattern hasresulted in a plethora of new β-lactamases spread-ing in Gram-negative pathogens, which reflectsboth the remarkable evolutionary potential of indi-vidual enzymes at the structural and functionallevel (likely facilitated by the lack of additionalfunctions essential to cell biology) and the greatstructural and functional diversity of the naturalβ-lactamase gene pool.

As such, the recent evolution of β-lactamase-mediated resistance in Gram-negative pathogensrepresents a paradigmatic example of how rapidand complex bacterial evolution can be under arapidly changing selective environment such as thatencountered in the clinical setting.

Future perspectiveThe fact that β-lactamase-mediated resistance canevolve following diverse and complex pathways, andthe presence of additional factors that influence bac-terial evolution at the population level (which areonly partially understood), make it very difficult topredict the future evolution of β-lactamase-medi-ated resistance. For instance, a few years ago it



would have been reasonable to predict a verymajor role for the TEM- and SHV-type ESBLs inthe future evolution of resistance to expanded-spectrum cephalosporins among Enterobacte-riaceae, while the CTX-M-type ESBLs, whichemerged at a later stage, are now replacing theTEM- and SHV-type ESBLs and rapidly chang-ing the ESBL epidemiology in several areas. Con-tinuous and capillary epidemiological surveillanceof these resistance determinants is essential tomonitor the evolution of the phenomenon,which in turn is important to develop controlstrategies and new therapeutic alternatives.

However, for the same reasons, it appears rea-sonable to foresee that β-lactamases will con-tinue to be an important mechanism ofβ-lactam resistance in Gram-negative patho-gens, and that new β-lactamases would likelyevolve against new β-lactams that are stable tothe currently circulating enzymes. In this per-spective, although inhibitor-resistant β-lacta-mases have also evolved, the development ofnew and potent inhibitors for the new β-lacta-mases would appear a valuable additional strat-egy to counteract the rapid evolution ofβ-lactamase-mediated resistance.

Executive summary

β-lactamases as drug resistance determinants• Among pathogenic bacteria, production of β-lactamases is one of the most important mechanisms of resistance to β-lactam

antibiotics. The introduction of new classes of β-lactams into clinical practice has invariably been followed by the emergence of new β-lactamases active on them. Evolution of β-lactamases in the clinical setting is a paradigmatic example of rapid bacterial evolution under a rapidly changing selective environment.

Clinically relevant β-lactamases at the beginning of the new century

• Currently, the β-lactamases of greatest clinical relevance are those capable of hydrolyzing the expanded-spectrum cephalosporins and the carbapenems, emerging among major Gram-negative pathogens such as Enterobacteriaceae, Pseudomonas aeruginosa and Acinetobacter. Based on some commonalities in functional/structural features, these enzymes can be included in three major groups: the extended-spectrum β-lactamases, the AmpC-type β-lactamases and the carbapenemases.

Mechanisms & complexity of evolution of β-lactamase-mediated resistance

• The evolution of β-lactamase-mediated resistance in major Gram-negative pathogens has followed multiple strategies including: mutational over-expression of resident enzymes, modulation of the activity of existing enzymes by mutation and capture of new β-lactamase genes from the natural β-lactamase gene pool by horizontal gene transfer. This complex evolutionary pattern, coupled with the remarkable evolutionary potential of individual enzymes and with the diversity of the natural β-lactamase gene pool, has resulted in a plethora of new β-lactamases spreading in Gram-negative pathogens.

Future perspective

• Prediction of the future evolution of β-lactamase-mediated resistance is very difficult, due to the diverse and complex evolutionary pathways involved. Continuous and capillary epidemiological surveillance is essential to monitor the evolution of the phenomenon. However, it is reasonable to foresee that β-lactamases will continue to be an important mechanism of β-lactam resistance, and that new β-lactamases would likely evolve against new classes β-lactams.

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Website101. Lahey Clinical

www.lahey.org/Studies• Comprehensive and updated web resource

for nomenclature and properties of clinically relevant β-lactamases.

Affiliations• Gian Maria Rossolini

University of Siena, Department of Molecular Biology, Section of Microbiology, Policlinico Santa Maria alle Scotte, 53100-Siena, ItalyTel.: +39 057 723 3455;Fax: +39 057 723 3870;[email protected]

• Jean-Denis DocquierUniversity of Siena, Department of Molecular Biology, Section of Microbiology, Viale Bracci, 53100-Siena, Italy and, University of Liège, Centre for Protein Engineering & Laboratory of Enzymology, Allée de la Chimie, Sart-Tilman, 4000-Liège, BelgiumTel.: +39 057 723 3134;Fax: +39 057 723 3870;[email protected]

new β-lactamases: a paradigm for the rapid response of bacterial evolution in the clinical setting

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