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Johnson & Johnson Pharmaceutical Research & Development, L.L.C., Raritan, New Jersey 08869
SUMMARY INTRODUCTION CLASSIFICATION SCHEMES MOLECULAR CLASS A CARBAPENEMASES Chromosomally Encoded Enzymes: SME, NMC, and IMI Plasmid-Encoded Enzymes: KPC and GES CLASS B METALLO-ß-LACTAMASES Recent Epidemiology CLASS D SERINE-CARBAPENEMASES: THE OXA ß-LACTAMASES DETECTION OF CARBAPENEMASES MICs Microbiological Tests with Inhibitors Biochemical and Molecular Tests CARBAPENEMASE ORIGINS AND TRANSMISSION CONCLUDING REMARKS REFERENCES
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| INTRODUCTION |
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Carbapenemases belong to two major molecular families, distinguished by the hydrolytic mechanism at the active site. The first carbapenemases described were from gram-positive bacilli. Unlike other ß-lactamases known at that time, these enzymes were inhibited by EDTA, thereby establishing them as metalloenzymes. Later work has shown that all metallo-carbapenemases contain at least one zinc atom at the active site that serves to facilitate hydrolysis of a bicyclic ß-lactam ring (46). In the mid- to late 1980s, another set of carbapenem-hydrolyzing enzymes emerged among the Enterobacteriaceae (134), but EDTA did not inhibit their activity (183). Subsequent studies showed that these enzymes utilized serine at their active sites and were inactivated by the ß-lactamase inhibitors clavulanic acid and tazobactam (183, 243).
Until the early 1990s, all carbapenemases were described as species-specific, chromosomally encoded ß-lactamases, each with a well-defined set of characteristics. However, the identification of plasmid-encoded IMP-1, a metallo-ß-lactamase in Pseudomonas aeruginosa (228), ARI-1 (OXA-23), a class D carbapenemase in Acinetobacter baumannii (157, 191), and KPC-1, a class A carbapenemase in Klebsiella pneumoniae (245), has changed the patterns of carbapenemase dissemination. What was once considered to be a problem of clonal spread has now become a global problem of interspecies dispersion. Because of the proliferation of new members of established carbapenemase families, it is even more important to try to understand the properties of these enzymes, with all their strengths and limitations. Several excellent carbapenemase reviews have appeared recently, including detailed compilations of the kinetic characteristics of these enzymes (112, 150, 221, 225). Therefore, this review focuses on updated information on the epidemiological and biochemical characteristics of both metallo- and serine carbapenemases.
| CLASSIFICATION SCHEMES |
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Classification based on amino acid homology has resulted in four major classes (5, 76, 84), which correlate well with the functional scheme but lack the detail concerning the enzymatic activity of the enzyme. Molecular classes A, C, and D include the ß-lactamases with serine at their active site, whereas molecular class B ß-lactamases are all metalloenzymes with an active-site zinc. Carbapenemases, ß-lactamases with catalytic efficiencies for carbapenem hydrolysis, resulting in elevated carbapenem MICs, include enzymes from classes A, B, and D.
Functional classification schemes that included carbapenemases were first proposed by Bush in 1988 (21). A number of subclassification schemes for the metallo-ß-lactamases have subsequently been proposed over the past 10 years. Rasmussen and Bush in 1997 suggested that the functional group 3 metalloenzymes (22) could be divided into three functional subgroups, based primarily on substrate specificities (182), whereas molecular subclasses have been proposed by Frere and colleagues for a number of years (46, 50, 51). In addition to these proposals, other modifications have been suggested for the classification of the metallo-ß-lactamases (60). These enzymes are currently divided into three subclasses based on a combination of structural features, zinc affinities for the two binding sites, and hydrolysis characteristics. A consensus scheme is shown in Table 1. Subclasses B1 and B3, divided by amino acid homology, bind two zinc atoms for optimal hydrolysis, while enzymes in subclass B2 are inhibited when a second zinc is bound. Subclass B2 also differs in hydrolysis spectrum, as it preferentially hydrolyzes carbapenems, in contrast to the broad hydrolysis spectrum observed for B1 and B3 enzymes (46).
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| MOLECULAR CLASS A CARBAPENEMASES |
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4 µg/ml) to fully resistant. These ß-lactamases, therefore, may go unrecognized following routine susceptibility testing. Three major families of class A serine carbapenemases include the NMC/IMI, SME, and KPC enzymes. Their hydrolytic mechanism requires an active-site serine at position 70 in the Ambler numbering system for class A ß-lactamases (6). All have the ability to hydrolyze a broad variety of ß-lactams, including carbapenems, cephalosporins, penicillins, and aztreonam, and all are inhibited by clavulanate and tazobactam, placing them in the group 2f functional subgroup of ß-lactamases. A fourth member of this class, the GES ß-lactamases, was originally identified as an ESBL family, but over time variants were discovered that had low, but measurable, imipenem hydrolysis. This subgroup of GES enzymes is also classified as functional group 2f carbapenemases. Table 2 shows the geographical and chronological detection of the class A carbapenemases.
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The IMI (for "imipenem-hydrolyzing ß-lactamase") and NMC-A (for "not metalloenzyme carbapenemase") enzymes have been detected in rare clinical isolates of E. cloacae in the United States, France, and Argentina (149, 175, 181, 183). NMC-A and IMI-1 have 97% amino acid identity and are related to SME-1, with approximately 70% amino acid identity (146, 183). They all contain the conserved active-site motifs S-X-X-K, S-D-N, and K-T-G of the class A ß-lactamases. In addition, these carbapenemases have conserved cysteine residues at positions 69 and 238 that form a disulfide bridge. The genes for these three ß-lactamases are all chromosomally located, with no evidence of mobile element association, a fact that may have contributed to their rarity. More recently, however, genes encoding IMI-2 ß-lactamases were found on plasmids in Enterobacter asburiae from United States rivers and on a plasmid from an E. cloacae isolate from China (9, 249).
Biochemical characterization of purified SME, NMC, and IMI enzymes revealed a broad hydrolysis spectrum that includes the penicillins, early cephalosporins, aztreonam, and carbapenems (Tables 3 and 4). Imipenem hydrolysis was easily measurable, with kcat values of >30 s–1. Meropenem had lower kcat and Km values than did imipenem. Cefoxitin and extended-spectrum cephalosporins were inefficiently hydrolyzed, when hydrolysis could be detected at all, and cefotaxime was hydrolyzed faster than ceftazidime (124, 179, 183).
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Two crystal structures of NMC-A, one native and the other complexed with a penicillanic acid inhibitor, and a structure for SME-1 all show that the overall structure and catalytic residues are similar to those of other class A ß-lactamases (139, 199, 201). For NMC-A, the structure of the enzyme did not change conformation when the inhibitor was bound. The disulfide bond between positions 69 and 238 is located near the active site, a feature common among the class A carbapenemases. This disulfide bond is necessary for hydrolytic activity, not just for imipenem hydrolysis, suggesting that it is required to stabilize the enzyme structurally (123, 199).
The two most prominent distinctions between the class A TEM-1 structure and NMC-A are a 1-Å alteration of the position of Asn132, which enlarges the substrate-binding cavity, and a change in conformation of the S3 strand between residues 237 and 240, which increases access to the active site. However, in the crystal structure of SME-1, the position of Asn132 was almost identical to the Asn132 residue position of TEM-1 (199). The major difference in the SME-1 structure compared to TEM and NMC-A was crowding of Ser70 and Glu166 in the active-site cleft, leaving no room for a catalytic water molecule. It is possible that conformational rearrangements upon substrate binding may allow a catalytic water molecule into this position. The structure of SME-1 complexed with a ß-lactam molecule would test this hypothesis.
In structure/function studies using site-directed mutagenesis, the ability of the SME-1 ß-lactamase to hydrolyze imipenem could not be attributed to a single amino acid residue (122). A substitution of alanine for serine at position 237 reduced imipenem kcat values from 100 s–1 to 20 s–1; hydrolysis of cephalothin was also reduced fivefold, but the benzylpenicillin kcat values remained unchanged (200). It seems likely that the important conserved residues of the group 2f carbapenemases together contribute to form an active site that can accommodate and hydrolyze the carbapenems.
The first member of the KPC family was discovered through the ICARE surveillance project in a K. pneumoniae clinical isolate from North Carolina in 1996 (245). This isolate was resistant to all ß-lactams tested, but carbapenem MICs decreased in the presence of clavulanic acid. Carbapenemase activity, first detected with a bioassay, was associated with a large plasmid that encoded the KPC-1 ß-lactamase.
The discovery of KPC-1 was quickly followed by several reports of a single-amino-acid variant, KPC-2, along the east coast of the United States (137, 138, 246). KPC-2 was first identified in 2003 as the result of a point mutation in KPC-1 and appeared in four isolates with imipenem MICs of 2 to 8 µg/ml from Baltimore, MD, from 1998 to 1999. The KPC-2-producing gene resided on a transferable plasmid, and it was noted that while all isolates exhibited reduced susceptibility to imipenem, none were technically resistant according to approved CLSI (formerly NCCLS) breakpoints (138). KPC-2 was then described in another Maryland site on a plasmid in Salmonella enterica (137). Genetic regions around this KPC-2 gene contained three open reading frames with sequence homology to transposases.
Reports of KPC-2 in the New York, NY, area began to appear in 2004, with KPC-expressing K. pneumoniae currently an alarming problem (13, 15, 17). This is especially disturbing because New York has had large outbreaks of ESBL-producing klebsiellae (135, 177) for which carbapenems were considered to be one of the few treatment options (156). When ribotyping was conducted on the KPC-2-producing strains, the majority of the isolates were clonal, even when the surveillance included multiple hospitals in the New York metropolitan area. Notably, several of these reports described inconsistencies in recognizing some of these strains as carbapenem resistant, because carbapenem MICs were less than the approved MIC breakpoints (13, 15, 16, 118).
Concurrent with the increasing reports of KPC-2, a single-amino-acid variant of KPC-2, KPC-3, was reported from a 2000 to 2001 Klebsiella pneumoniae outbreak in New York (233). KPC-3 has also been detected in Enterobacter spp. (14), where MICs for imipenem were also not consistently in the resistant range. Kinetic analysis of the KPC-3 enzyme revealed a profile similar to those of KPC-1 and KPC-2, with a slight increase in the hydrolysis of ceftazidime (4).
After the rapid expansion of the KPC class of carbapenemases along the east coast of the United States, worldwide reports began to appear. A report from France in 2005 documented KPC-2 in a K. pneumoniae strain from a patient who had been in New York for medical treatment (145). KPC carbapenemases have recently been detected in Scotland (KPC-4 [GenBank accession no. AY700571]), Colombia (217), Israel (147), and China (229). The first detection of KPC-2 on a plasmid in P. aeruginosa has been reported; this represents a disturbing development in the spread of these carbapenemases (216).
KPC enzymes have the conserved active-site motifs S-X-X-K, S-D-N, and K-T-G of the class A ß-lactamases and have the closest amino acid identity (
45%) to the SME carbapenemases. In addition, these ß-lactamases have the conserved C69 and C238 residues that form a disulfide bond described for the SME and NMC/IMI enzymes. The structure of the KPC-2 ß-lactamase, compared to the SME-1 and NMC-A carbapenemases and the TEM-1 and SHV-1 noncarbapenemases, reveals characteristics conserved among the carbapenemases. KPC-2, along with the other carbapenemases, had a decrease in the size of the water pocket and had the catalytic serine in a more shallow position of the active-site cleft (92). The combination of subtle active-site adjustments in the class A carbapenemases is proposed to allow carbapenems access to the catalytic site, resulting in the altered specificity of these enzymes.
KPC carbapenemases hydrolyze ß-lactams of all classes, with the most efficient hydrolysis observed for nitrocefin, cephalothin, cephaloridine, benzylpenicillin, ampicillin, and piperacillin (Tables 3 and 4). Imipenem and meropenem, as well as cefotaxime and aztreonam, were hydrolyzed 10-fold-less efficiently than the penicillins and early cephalosporins. Weak but measurable hydrolysis was observed for cefoxitin and ceftazidime, giving the KPC family a broad hydrolysis spectrum that includes most ß-lactam antibiotics.
Of the functional group 2f carbapenemases, the KPC family has the greatest potential for spread due to its location on plasmids, especially since it is most frequently found in K. pneumoniae, an organism notorious for its ability to accumulate and transfer resistance determinants. In addition, the clonal spread seen in several epidemics points to difficulties with infection control for this organism (13, 15-17, 233). Most worrisome, treatment of infections caused by these organisms is extremely difficult because of their multidrug resistance, which results in high mortality rates (15).
The GES/IBC family of ß-lactamases is an infrequently encountered family that was first described in 2000 with reports of IBC-1 (for "integron-borne cephalosporinase") from an E. cloacae isolate in Greece (53) and GES-1 (for "Guiana extended spectrum") in a K. pneumoniae isolate from French Guiana (168). These enzymes differ by only two amino acid substitutions and possess the class A active site-motifs with the cysteine residues at Ambler positions 69 and 238 that have been found in the KPC, SME, and NMC/IMI families. Their amino acid sequences show them to be distantly related to these carbapenemases, with identities of 36% to KPC-2, 35% to SME-1, and 31% to NMC-A (174).
The genes encoding the GES family of enzymes were located in integrons on plasmids. Because the enzymes had a broad hydrolysis spectrum that included penicillins and extended-spectrum cephalosporins, they were initially classified as extended-spectrum ß-lactamases (53). Their hydrolysis spectrum was expanded in 2001 to include imipenem, with the report of GES-2 in a clinical isolate of P. aeruginosa (174). GES-2, from a multidrug-resistant P. aeruginosa isolate from South Africa, had a single amino acid substitution of glycine to asparagine at position 170. Imipenem hydrolysis by GES enzymes was slow, with hydrolytic rates of 0.004 s–1and 0.004 s–1 for GES-1 and GES-2, respectively. However, the hydrolytic efficiency for imipenem was 100-fold higher for GES-2, due to a 100-fold decrease in the Km value. The GES-4 ß-lactamase differed from GES-2 by three amino acids, one of which was a serine at position 170. Carbapenem kcat values for purified GES-4 were higher than those for GES-2, with imipenem hydrolyzed at a rate of 0.38 s–1 (219).
Nomenclature of the GES/IBC family has undergone several revisions (105). A consensus nomenclature has been reached whereby the IBC names have been converted to the GES nomenclature (83, 105). At least nine GES variants have been described, with GES-9 recently identified in a P. aeruginosa isolate from France (164). Of these closely related enzymes, GES-2, GES-4, GES-5, and GES-6 have substitutions of asparagine or serine at position 170, associated with imipenem hydrolysis (105, 218).
Although rare, GES enzymes have been identified worldwide, with reports from Greece, France, Portugal, South Africa, French Guiana, Brazil, Argentina, Korea, and Japan (25, 33, 42, 86, 90, 155, 164, 168, 173, 186, 218-220). These enzymes have been most frequently associated with single occurrences. However, P. aeruginosa strains expressing GES-2 have caused a small nosocomial outbreak in eight patients (173), and six patients in Korea had infections caused by GES-5-producing K. pneumoniae (86).
| CLASS B METALLO-ß-LACTAMASES |
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The first metallo-ß-lactamases detected and studied were chromosomal enzymes present in environmental and opportunistic pathogenic bacteria such as Bacillus cereus (98, 109), Aeromonas spp. (78), and Stenotrophomonas maltophilia (188). These chromosomal enzymes were usually found in bacteria that also expressed at least one serine ß-lactamase, with both ß-lactamases inducible after exposure to ß-lactams. Fortunately, with the exception of S. maltophilia, these bacteria have not been frequently associated with serious nosocomial infections, as they are generally opportunistic pathogens, and the chromosomal metallo-ß-lactamase genes are not easily transferred.
Early classification based on functional analyses of purified proteins indicated that these ß-lactamases were distinctive from the groups of enzymes that had a serine-based hydrolytic mechanism (21). Major distinctive properties included the requirement of Zn2+ for the efficient hydrolysis of ß-lactams and a lack of inhibition by clavulanic acid and tazobactam. A defining aspect of their substrate spectrum was the ability to hydrolyze carbapenems (Table 4). Interestingly, not all of the metallo-ß-lactamases readily hydrolyzed nitrocefin, the popular colorimetric indicator of ß-lactamase activity (130). The "hidden" carbapenemases of Aeromonas spp. hydrolyzed nitrocefin and other cephalosporins imperceptibly and could be detected only by using a carbapenem bioassay or hydrolysis test (64, 65, 196, 223, 241). Recently, the VIM-2 enzyme was shown to share this property and was detected on IEF gels by an imipenem hydrolysis detection method (171).
Since the initial identification of the metallo-ß-lactamases, a considerable amount of sequencing work has demonstrated high variability in primary sequences and in molecular structures. The first metallo-ß-lactamases for which an amino acid sequence was determined was the BCII metallo-ß-lactamase from Bacillus cereus (77), the prototypical metallo-ß-lactamase for many years. Extensive molecular characterization, including biochemical and crystallization studies, has also been completed with the CcrA (CfiA) chromosomal metallo-ß-lactamase that appears in a small percentage of Bacteroides fragilis strains (32, 242). Amino acid sequence identities are as low as 23% across the metallo-ß-lactamases, including enzymes such as CcrA, CphA, and L1 (Table 1) (182, 224). However, all of the enzymes have conserved residues that bind zinc at two sites (see "Classification Schemes," above, and Table 1) and exhibit a well-conserved active site, as demonstrated through sophisticated modeling analyses (127) and in the crystal structures published to date (31, 32, 34, 44, 52, 141, 214).
In contrast to the chromosomal metallo-ß-lactamases, whose presence is directly correlated with the prevalence of the producing species, there has been a dramatic increase in the detection and spread of the acquired or transferable families of these metalloenzymes. The most common metallo-ß-lactamase families include the VIM, IMP, GIM, and SIM enzymes, which are located within a variety of integron structures, where they have been incorporated as gene cassettes. When these integrons become associated with plasmids or transposons, transfer between bacteria is readily facilitated.
Transferable imipenem resistance was first detected in Japan, initially in a P. aeruginosa isolate, in 1990 (228), followed by a second report of a transferable carbapenemase in B. fragilis (11). The transferable B. fragilis enzyme was one of the first characterized metallo-ß-lactamases, but this species has not caused widespread clinical outbreaks in Japan. In contrast, IMP-1 (for "active on imipenem"), located on a conjugative plasmid in the P. aeruginosa clinical isolate (228), was found on an integron in S. marcescens and other Enterobacteriaceae in Japan (7, 72, 80, 195). This enzyme hydrolyzed imipenem, penicillins, and extended-spectrum cephalosporins but not aztreonam. The hydrolytic activity was inhibited by EDTA and restored by the addition of Zn2+. The first member of the IMP family found in Europe was in an A. baumannii isolate from Italy, which produced a related enzyme, IMP-2, as the first cassette on a class 1 integron (184). Since that time, the IMP family has been found throughout the world, with the most recent spread to the United States and Australia (62, 159). The historical discovery of IMP-type ß-lactamases is well documented in the recent review by Walsh et al. (224). Currently, the IMP family members number up to 18 in the published literature, with 23 assigned IMP sequences listed on the ß-lactamase nomenclature website (http://www.lahey.org/Studies/ [updated 17 November 2006]).
Another prevalent family of integron-associated metallo-ß-lactamases is composed of the VIM enzymes. VIM-1 (for "Verona integron-encoded metallo-ß-lactamase") was first isolated in Verona, Italy, in 1997 (101), with the identification of VIM-2 in France in 1996 subsequently reported (171). Both of these enzymes were initially found in P. aeruginosa clinical isolates and resided in class 1 integrons. The VIM family currently consists of 14 members (http://www.lahey.org/Studies/ [updated 17 November 2006]), with occurrences mostly in P. aeruginosa within multiple-integron cassette structures. VIM-2 has the dubious distinction of being the most-reported metallo-ß-lactamase worldwide (224).
Identification of the SPM-1 metallo-ß-lactamase defined a new family with 35.5% amino acid identity to IMP-1 (206). SPM-1 (for "Sao Paulo metallo-ß-lactamase") was first isolated in a P. aeruginosa strain in Sao Paolo, Brazil. Since the initial report, single clones of SPM-1-containing P. aeruginosa have caused multiple hospital outbreaks with high mortality in Brazil (126, 169, 251). Genetic analysis of regions around the SPM-1 gene revealed that it was not part of an integron but instead was associated with common regions that contain a new type of transposable structure with potential recombinase and promoter sequences (169, 206).
GIM-1 (for "German imipenemase") was isolated in Germany in 2002 (26). GIM had approximately 30% homology to VIM, 43% homology to IMPs, and 29% homology to SPM. GIM-1 has characteristics similar to those of the other acquired metallo-ß-lactamases in that it was found in five clonal P. aeruginosa isolates within a class 1 integron on a plasmid. At this time, it has not been reported elsewhere in the world.
The latest family of acquired metallo-ß-lactamases to be described comes from Korea. The enzyme SIM-1 (for "Seoul imipenemase") has the closest amino acid identity to the IMP family (64 to 69%). SIM-1 was discovered in a large-scale screen of 1,234 imipenem-resistant Pseudomonas sp. and Acinetobacter sp. isolates, of which 211 (17%) were positive for metallo-ß-lactamases. In this screening study, mostly VIM (74%) and IMP (22%) alleles were identified; however, seven putative metallo-ß-lactamase-producing A. baumannii isolates were negative by PCR testing. Investigation with PCR primers designed to amplify entire integrons revealed that they had the novel SIM-1 metallo-ß-lactamase located within a class 1 integron (104). Two clonal groups, of four and three strains, carried the same integron, suggesting independent acquisition events.
Since their initial discoveries, SPM, GIM, and SIM metallo-ß-lactamases have not spread beyond their countries of origin. However, VIM and IMP continue to be detected worldwide, with an overall trend of these two metallo-ß-lactamases moving beyond P. aeruginosa and into the Enterobacteriaceae (Table 5).
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In a recent survey from Korea of 15,960 gram-negative clinical isolates (247), metallo-ß-lactamases were found in 36 of 581 imipenem-resistant isolates. In P. aeruginosa, VIM-2-like enzymes were the majority of metallo-ß-lactamases in this sample; however, two strains tested positive by PCR for IMP-1-like enzymes. For the Acinetobacter species, 26.5% (136 of 513) of the imipenem-resistant strains carried metallo-ß-lactamases: 64% were VIM-2-like, 29% were IMP-1-like, and 7% were SIM-like. Among the Enterobacteriaceae, resistance to imipenem was 2% for K. pneumoniae and <1% for E. cloacae and S. marcescens, which tested positive by PCR for VIM-2.
IMP metallo-ß-lactamases continue to persist as the major metalloenzymes found in Japan, both at a local (148) and a country-wide (94) level. However, VIM-2-like genes have also been detected in P. aeruginosa. IMP-1 and IMP-2-like enzymes were found in Acinetobacter spp., as well as S. marcescens, P. rettgeri, C. freundii, E. cloacae, and M. morganii. Among almost 20,000 clinical gram-negative isolates from 13 clinical laboratories, the overall rate of metallo-ß-lactamase detection was 0.5% in Japan. In these reports, there was as much as 2.6% metallo-ß-lactamase detection in imipenem-resistant P. aeruginosa; S. marcescens had the highest rate of IMP gene detection in Japan, at 3%. In a SENTRY surveillance study conducted in Japan, 1.1% of the P. aeruginosa strains expressed metallo-ß-lactamases (88); among these, IMP alleles were the only metallo-ß-lactamases detected.
The first metallo-ß-lactamase reported from China was IMP-4, which was found on a plasmid in C. youngae and reported in a 2001 publication (63), followed by a report of IMP-1 in a P. aeruginosa isolate (226). Recently, IMP-9 has emerged in this country, as reported in a surveillance study that included 11 hospitals (234). The IMP-9 genes were all on identical integrons, and only two strains that came from the same ward were related, based on random amplified polymorphic DNA typing. A very large plasmid (450 kb) was shown to carry the IMP-9 gene by conjugation experiments, suggesting that the spread of this IMP-containing integron was through horizontal transfer.
The VIM-2 metallo-ß-lactamase was first reported in China only in 2006 (227). This single P. aeruginosa isolate occurred in the same hospital as the IMP-1 isolate (226). At this point, the route for emergence of these enzymes is unknown, but further epidemiological analysis, sequencing of the integrons, and typing of the strains could answer questions about whether the strains imported or acquired metallo-ß-lactamases independently.
In Taiwan, IMP-1 was reported in two A. baumannii isolates that were unrelated by PFGE. In these isolates, the IMP-1 gene was on two different plasmids within class 1 integrons (111). VIM-2 and VIM-3 have also been detected within integrons in multidrug-resistant P. aeruginosa (236).
In southern Europe, particularly in Italy, there has been almost continuous detection of VIM and IMP metallo-ß-lactamases throughout many countries over the past decade. In Italy, a recent SENTRY study for the years 2001 and 2002 found that 25 of 383 (6.5%) P. aeruginosa isolates from three medical centers carried metallo-ß-lactamases (88). The majority of metallo-ß-lactamases identified were VIM-1, but IMP-13 was also detected. Some of the VIM-1-expressing isolates from Rome and Catania carried the integron described in the original VIM-1 from Verona; differences in ribotypes demonstrated the mobility of this integron. Several unique integron cassette arrangements were reported, including some strains possessing as many as four integrons, thus leaving open the possibility for more integron gene rearrangements (100). In another study reported by Pagani et al., IMP-13 was responsible for a large clonal outbreak of at least 86 metallo-ß-lactamase-producing P. aeruginosa strains in southern Italy (154). The integron, which was the same as the IMP-13 found in the SENTRY study, was localized to the chromosome and not transferable to P. aeruginosa or Escherichia coli by conjugation. Another surveillance study of 506 P. aeruginosa isolates from a single center in Varese showed 82% IPM susceptibility, but with only four strains carrying metallo-ß-lactamases, (VIM-1 and VIM-2), demonstrating the regional variation in resistance patterns due to these enzymes (121).
More frequent reports of metallo-ß-lactamases are now being published from other European countries, documenting further detections of acquired carbapenem resistance. P. aeruginosa isolates with integron-associated VIM alleles have been reported in Germany (66), Turkey (10), Croatia (190), Hungary (107, 108), and Poland (45).
Metallo-ß-lactamase outbreaks in South America are dominated by the SPM and VIM families. In a recent SENTRY study of 183 P. aeruginosa isolates, 44.8% were imipenem resistant; of these, 36 isolates were positive for metallo-ß-lactamases. The majority of the isolates were positive for SPM-1-like genes by PCR, (55.6%), followed by VIM-2-type (30.6%) and IMP-1-like genes in three isolates (8.3%) (88, 187). While SPM-1 has been confined to Brazil, other countries in South America that have reported metallo-ß-lactamases are Argentina, Chile, and Venezuela (88), as well as Colombia, where both VIM-2 and VIM-8 were found in P. aeruginosa (36, 217).
Emergence of metallo-ß-lactamase-mediated carbapenem resistance has spread to the United States and Canada, with reports of both VIM and IMP metallo-ß-lactamases in P. aeruginosa. VIM-7 was isolated in Texas in 2001 from a single strain that carried the integron on a conjugative plasmid (204). More recently, VIM-2 has also been detected in Texas (1). VIM-2 was also found in Chicago, IL, in an outbreak setting of clonal P. aeruginosa involving 17 patients admitted from 2002 to 2004 (115), where the metallo-ß-lactamase was present on an integron. Metallo-ß-lactamases have also emerged as outbreaks of VIM-2- and IMP-7-producing P. aeruginosa in Canada (55, 163) and in a single P. aeruginosa isolate from the southwestern United States that produced IMP-18 (62).
Australia has also reported detection of metallo-ß-lactamases. The first metalloenzyme that emerged on the Australian continent was IMP-4, reported in 2004 in a single P. aeruginosa clinical isolate (159) and in single K. pneumoniae and E. coli isolates (172), followed by an outbreak of IMP-4-producing gram-negative pathogens (158). Recently, an Aeromonas junii isolate from Australia was found to have two carbapenemases: a class D OXA-58 and an IMP-4 metallo-ß-lactamase (160).
Other areas of the world that have recently reported metallo-ß-lactamases include the Middle East, where VIM-2 was the first reported metallo-ß-lactamase. It was detected by PCR in an imipenem-resistant P. aeruginosa isolate from Saudi Arabia (58). Yong et al. recently reported the detection of metallo-ß-lactamases from India by using an EDTA disk test (248), where 13 of 200 (7.5%) Pseudomonas sp. and Acinetobacter sp. isolates were positive for the production of metallo-ß-lactamases. Although further analysis of these enzymes is not yet available, awareness and early detection may prevent uncontrolled outbreaks due to these pathogens (59).
| CLASS D SERINE-CARBAPENEMASES: THE OXA ß-LACTAMASES |
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The vast majority of OXA carbapenemases have been discovered in the opportunistic gram-negative pathogen Acinetobacter baumannii. By 1998, carbapenem-hydrolyzing ß-lactamases had been identified in Acinetobacter species clinical isolates throughout the world (2). For example, OXA-23 has been identified in outbreaks of carbapenem-resistant Acinetobacter in Brazil, the United Kingdom, Korea, and Tahiti (37, 85, 142, 211). OXA-24 and OXA-40, which differ by two amino acids, were found in clonal Acinetobacter outbreaks in hospitals from Spain and Portugal (12, 40, 119). OXA-40 was also the first carbapenem-hydrolyzing oxacillinase reported in the United States (116). Acinetobacter strains with OXA-23 and OXA-58 carbapenemases caused multiple infections in military and civilian personnel serving in Iraq and Afghanistan from 2003 to 2005 (75).
At the latest count, there are nine major subgroups of OXA carbapenemases, based on amino acid homologies, as summarized in Table 6 (18, 167, 225). Subgroups 1, 2, and 3 are based on the sequences of OXA-23, OXA-24, and OXA-51, respectively (213). OXA-51-like enzymes have been found in all A. baumannii strains tested and may be a natural component of the chromosome in a subpopulation of that species (69). OXA-58, less that 50% identical to other members of the OXA family, stands alone in subgroup 4 and has been found in Acinetobacter spp. from France, Greece, Italy, Romania, Turkey, Argentina, and Kuwait (28, 68, 125, 170, 215). OXA-55 and OXA-SHE, both from Shewanella algae, form the fifth group (71). The OXA-48 enzyme forms the sixth subgroup, along with OXA-54 and additional oxacillinases found in the environmental bacteria Shewanella spp. (165-167). In contrast to the sharp increase in worldwide reports of OXA-expressing Acinetobacter strains, OXA-48 was discovered in a clinical K. pneumoniae isolate from Turkey (167). This OXA variant was plasmid encoded and had less than 50% amino acid identity to the other OXA members. This enzyme also had the highest reported imipenem kcat value, 2 s–1, which represents the highest hydrolysis rate of all of the published kinetic parameters for the OXA enzymes.
The OXA-50-like enzymes in P. aeruginosa form the seventh group and include a set of enzymes that have been referred to as the poxB enzymes. These PoxB oxacillinases have been reported to be commonly present on the chromosomes of many strains of P. aeruginosa (40 of 70 strains tested) (96) and are thought to be part of the natural component of ß-lactamases in that species, but they may not be expressed in all strains and do not cause carbapenem resistance (imipenem MIC, 1 µg/ml) (56, 96). Species-specific OXA enzymes include the subgroup 8 OXA-60 family, considered to be a natural component of the genome of Ralstonia pickettii (57), and the subgroup 9 OXA-62, identified as a species-specific oxacillinase in Pandoraea pnomenusa (192).
The OXA ß-lactamases display a wide variety of amino acid sequences. Among those with carbapenem-hydrolyzing activity, there is 40% to 70% amino acid identity between groups. Within a group the identity is greater than or equal to 92.5% (225). If OXA ß-lactamases without carbapenem-hydrolyzing activity are considered, amino acid identities can be as low as 18%, for example, when OXA-1 is compared with OXA-58 (170).
The molecular structures of the OXA ß-lactamases have been analyzed with the DBL numbering system (35), a classification based on the similarity of motifs around the active sites of the class A enzymes (6). The catalytic serine residue lies in the S-T-F-K tetrad at positions 70 to 73 (102), where the serine and lysine are conserved in both class A and class D enzymes, as well as in penicillin-binding proteins. The Y-G-N motif at positions 144 to 146 and the K-T-G at DBL 216 to 218 are highly conserved among the serine-based ß-lactamases. The carbapenem-hydrolyzing OXA subgroups 1 and 2 share a substitution of F for Y in the Y-G-N motif, but this is not necessary for imipenem hydrolysis, as the subgroup 3 enzymes and OXA-58 retain the Y-G-N at this position.
The first crystal structure of the class D carbapenemases to appear in the literature was that of OXA-24, which was compared to the noncarbapenemase OXA-10 (189). Two amino acids, Tyr-112 and Met-223, make access to the active site smaller and more hydrophobic, allowing favorable interactions with carbapenems. These residues are found in other subgroups of OXA carbapenemases, suggesting that the altered active-site access is an important contributing factor to the carbapenem-hydrolyzing activity among these enzymes.
The catalytic mechanism of the OXA ß-lactamases shares features with other serine carbapenemases. Substrate and enzyme form a covalent acyl intermediate at the catalytic serine, which is subsequently deacylated to yield the inactivated antibiotic hydrolyzed at the C-N bond of the ß-lactam ring. In addition, CO2 may influence the kinetics of some OXA enzymes, due to carboxylation at lysine 70 (Lys 73 in the DBL scheme) in these class D enzymes. This carbamate activates the catalytic serine side chain so that the acyl intermediate can form (131, 153). To ensure that this modification occurs in studies with isolated enzymes, some researchers add 10 mM NaHCO3 to their reactions (57, 69, 192).
OXA enzymes are difficult to purify due to low yield (3) and difficult to characterize biochemically due to low hydrolysis rates and biphasic kinetics for some substrates. The OXA carbapenemases that have been characterized biochemically have measurable hydrolytic activity against the penicillins, some cephalosporins, and imipenem (225). In general, imipenem hydrolysis, which is faster than meropenem hydrolysis, is slow, with the highest kcat values for the group 6 OXA-54 and OXA-48 enzymes at 1 s–1 and 2 s–1, respectively. The Km values for imipenem are also low, ranging from 2 to 20 µM, indicating that the OXA enzymes have very high affinity for these substrates. In contrast, extended-spectrum cephalosporins are not measurably hydrolyzed by the OXA carbapenemases or are hydrolyzed very poorly (225).
The hydrolysis of carbapenems by the class D oxacillinase family is weak, but Heritier et al. (70) demonstrated that plasmid-encoded OXA-23 and OXA-58 enzymes contributed to carbapenem resistance in A. baumannii after transformation of the OXA plasmids into carbapenem-susceptible A. baumannii strains. In addition, when a chromosomally located OXA-40 gene was inactivated, susceptibility to carbapenems was observed. Efflux by an overexpressed AdeABC pump was also shown to contribute to carbapenem resistance.
| DETECTION OF CARBAPENEMASES |
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2 µg/ml (225). Elevated carbapenem MICs are generally predictive of carbapenemase production in the Enterobacteriaceae, but full clinical resistance is not always seen. In a set of 19 K. pneumoniae isolates with imipenem MICs in the susceptible range of 1 to 4 µg/ml, a metallo-ß-lactamase was suggested on the basis of disk testing with imipenem in the presence and absence of EDTA and was then confirmed by PCR as VIM-1 (161). A collection of five related K. pneumoniae strains with the VIM-1 gene demonstrated imipenem MICs ranging from 2 to 64 µg/ml (susceptible to high-level resistance) (117). Decreased permeability due to the absence of the outer membrane porin OmpK36 was a contributing factor in the most resistant isolate, with an imipenem MIC of 64 µg/ml.
The KPC serine carbapenemases also have been reported to be difficult to detect (14, 16, 138). They are often associated with imipenem MICs as low as 2 µg/ml (138), and a low inoculum has resulted in susceptible MICs by broth microdilution (15). To document inconsistencies in the detection of KPC-producing K. pneumoniae according to the testing method, Tenover et al. tested 15 characterized imipenem- and meropenem-nonsusceptible KPC-producing isolates for imipenem and meropenem resistance using CLSI broth microdilution, Etest, MicroScan WalkAway, BD Phoenix Sensititre Autoreader, VITEK, and VITEK2. The automated systems reported carbapenem susceptibility in this collection of isolates ranging from 6.7% to 87%, depending on the system used (203). Day-to-day variation was also noted. In addition to the variable results with the automated systems, Etest results were inconsistent due to colonies present in the zones of inhibition.
The failure of automated systems to consistently detect KPC-producing isolates indicates the need for improved methodology. One possibility is to screen isolates for resistance to ertapenem, which had the highest sensitivity for detecting KPC-expressing isolates (16). However, the specificity may be reduced due to resistance from other mechanisms, such as AmpC or ESBL production coupled with porin loss (N. Woodford, J. Dallow, R. Hill, M. F. Ralepou, R. Pike, M. Ward, M. Warner, and D. Livermore, presented at the 46th Interscience Conference on Antimicrobial Agents and Chemotherapy, San Francisco, CA, 27 to 30 September 2006).
The NMC and IMI serine carbapenemases in E. cloacae are associated with imipenem MICs of 16 to 32 µg/ml, with ceftazidime MICs often 2 µg/ml (149, 183). In S. marcescens, the SME family of ß-lactamases should be suspected if an isolate displays this pattern of high-level imipenem resistance associated with ceftazidime susceptibility.
Etest strips for metallo-ß-lactamase testing are also available as imipenem and imipenem-EDTA combinations (AB BIODISK, Solna, Sweden). A positive test for a metallo-ß-lactamase is interpreted as a threefold-or-greater decrease in the imipenem MIC in the presence of EDTA. This test strip produced a sensitivity of 94% and a specificity of 95% when examined with a set of 138 characterized metallo-ß-lactamase producers (222). However, false-negative results have been reported for the Etest when an isolate had an imipenem MIC of <4 µg/ml (222, 239). It has also been observed that EDTA alone has inhibitory action against some bacteria due to permeabilization of the outer membrane and can lead to false-positive results (27). Etest metallo-ß-lactamase detection tests have also yielded false-positive results with OXA-23-producing A. baumannii (194).
Carbapenem inactivation assays can be a fast, sensitive method for initial characterization of carbapenem-resistant isolates. The cloverleaf test is a microbiological assay of carbapenemase activity where suspensions of whole cells or and/or an extract of the suspect isolate are tested against imipenem on an agar plate (73). Altered growth of an indicator strain around an imipenem disk is a positive result. One advantage of this test is that enzymes that have very weak carbapenemase activity, such as OXA-23 (73) or GES-5 and GES-6 (218), can be detected by this method.
IEF can also be combined with a bioassay to detect the presence of carbapenemases by using an overlay of agar with imipenem and a second overlay with a susceptible indicator organism (103, 192, 241). Growth over an enzyme band indicates a potential carbapenemase. This procedure is especially useful when working with carbapenemases that have poor hydrolysis rates with nitrocefin, such as the metallo-ß-lactamases from Aeromonas spp. (241).
Imipenem hydrolysis can most reliably be detected with a spectrophotometric measurement using crude cell extracts or purified ß-lactamases (101). If the carbapenemase is a metalloenzyme, a brief incubation with EDTA prior to initiation of the reaction will result in a lower hydrolysis rate. Very weak carbapenemases cannot be detected by this method unless large amounts of extracts are used.
When the presence of a carbapenemase is suspected, PCR is the fastest way to determine which family of ß-lactamase is present. Table 7 lists a selection of published primers that have been used with standard PCR technology to detect all of the families and subgroups of carbapenemases known at this time.
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Ultimately, the identification of the ß-lactamase gene requires sequencing of the entire coding region. Cloning of the region around the ß-lactamase is usually accomplished with a "shotgun" approach, but a clever degenerate PCR method has also been used successfully to amplify 5' and 3' areas surrounding OXA-51 (19). Characterization of a new ß-lactamase is not complete until both a molecular sequence is obtained and a functional analysis of the hydrolysis and inhibition profiles is performed with purified protein.
| CARBAPENEMASE ORIGINS AND TRANSMISSION |
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Investigators looking for prospective sources of carbapenemase genes have been able to find several in environmental species. The class A carbapenemase SFC-1 was described in an environmental isolate of Serratia fonticola (67). Several of the OXA carbapenemase genes such as OXA-50 and its variants in P. aeruginosa (56, 96), OXA-51-like enzymes in A. baumannii (69, 213), OXA-62 in Pandoraea pnomenusa (192), and OXA-54 and OXA-55 in Shewanella spp. (71, 165) appear to be natural components of their respective bacterial chromosomes. In the case of the OXA genes in A. baumannii, insertion sequences of the ISAba1 type, carrying strong promoters, have been detected upstream of the chromosomal oxacillinase, resulting in increased expression and concomitant carbapenemase resistance (212). Transmission of carbapenemase genes is accelerated when the gene is located within mobile elements such as plasmids and integrons.
In addition to the finding of novel carbapenemases in environmental isolates, enzymes first detected in the clinic are now being found in environmental bacteria. The VIM-2 carbapenemase was found in a Pseudomonas pseudoalcaligenes strain from a hospital wastewater system (180); further examination found two P. aeruginosa strains with this gene. In another interesting study, bacteriophages carrying ß-lactamase genes for OXA-type ß-lactamases were isolated from sewage, suggesting another vector for transfer of these genes between organisms (140).
While it is not hard to imagine carbapenemase-producing strains finding their way into the sewage system, the discovery of IMI-2 carbapenemases on plasmids in rare E. asburiae isolates from U.S. rivers is harder to explain, although the location of sampling in relation to population centers was not reported (9). It is likely that the circulation of carbapenemase genes proceeds in two directions: environmental sources may provide genetic material as a source of these enzymes, and clinical strains may disperse this information both within the hospital setting and into the surrounding environment.
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