Extended-spectrum Enzymes Research Articles

BETA-LACTAMASES: PHENOTYPIC IDENTIFICATION METHODS

Today, β-lactam antibiotics are the most widely used. This is due to their low toxicity, wide spectrum of action and variety of drugs. Therefore, the bacterial resistance factor in the form of β-lactamase production is of particular interest. Bacteriological laboratories have classical methods for detecting β-lactamases. However, the combination of bacterial production of enzymes of different classes, together with the overexpression of some of them, causes difficulties in recognizing the production of extended-spectrum enzymes, which is critical for making clinical decisions. Currently known phenotypic detection methods for β-lactamases and extended-spectrum carbapenemases, when performed correctly, allow precise elucidation of the mechanism of resistance. However, these methods are very labor-intensive, difficult to implement and require highly qualified staff of bacteriological laboratories. Perhaps methods based on molecular diagnostic approaches, due to automation and standardization, high accuracy and specificity in identifying β-lactamase genes, reducing research time, and the ability to identify uncultivable or slowly growing microorganisms, could simplify the determination of mechanisms of antibiotic resistance in the future.

The molecular basis of β-lactamase production in Gram-negative bacteria from Saudi Arabia.

Resistance to β-lactams among Gram-negative bacteria is a worldwide issue. Increased prevalence of extended-spectrum β-lactamase (ESBL)-producers and the dissemination of carbapenem-resistance genes are particularly concerning. ESBL-producing strains are common in the Kingdom of Saudi Arabia, particularly among the Enterobacteriaceae, and carbapenem resistance is on the increase, especially among the non-fermenters. β-lactamase production is a major mechanism of resistance to these agents and although β-lactamase-producing strains have been documented in the Kingdom, relatively few reports characterized the molecular basis of this production. Nevertheless, available data suggest that CTX-M (CTX-M-15 in particular) is the predominant ESBL in the Enterobacteriaceae, with SHV also being prevalent in Klebsiella pneumoniae. Carbapenem resistance in the latter is mainly due to OXA-48 and NDM-1. In Pseudomonas aeruginosa, VEB-like enzymes are the most common ESBLs, and VIM is the prevalent metallo-β-lactamase. OXA-10 extended-spectrum enzymes are also frequent. PER and GES ESBLs have been reported in Acinetobacter baumannii, and oxacillinases (OXA-23 in particular) are the dominant carbapanamases in this species.

Importance of Position 170 in the Inhibition of GES-Type β-Lactamases by Clavulanic Acid

Bacterial resistance to β-lactam antibiotics (penicillins, cephalosporins, carbapenems, etc.) is commonly the result of the production of β-lactamases. The emergence of β-lactamases capable of turning over carbapenem antibiotics is of great concern, since these are often considered the last resort antibiotics in the treatment of life-threatening infections. β-Lactamases of the GES family are extended-spectrum enzymes that include members that have acquired carbapenemase activity through a single amino acid substitution at position 170. We investigated inhibition of the GES-1, -2, and -5 β-lactamases by the clinically important β-lactamase inhibitor clavulanic acid. While GES-1 and -5 are susceptible to inhibition by clavulanic acid, GES-2 shows the greatest susceptibility. This is the only variant to possess the canonical asparagine at position 170. The enzyme with asparagine, as opposed to glycine (GES-1) or serine (GES-5), then leads to a higher affinity for clavulanic acid (K(i) = 5 μM), a higher rate constant for inhibition, and a lower partition ratio (r ≈ 20). Asparagine at position 170 also results in the formation of stable complexes, such as a cross-linked species and a hydrated aldehyde. In contrast, serine at position 170 leads to formation of a long-lived trans-enamine species. These studies provide new insight into the importance of the residue at position 170 in determining the susceptibility of GES enzymes to clavulanic acid.

CTX-M and SHV-12 β-lactamases are the most common extended-spectrum enzymes in clinical isolates of Escherichia coli and Klebsiella pneumoniae collected from 3 university hospitals within Korea

Among the 443 clinical isolates of Escherichia coli and Klebsiella spp. collected between June and November 2003 from 3 university hospitals in Korea, 62 isolates were confirmed as extended-spectrum β-lactamase (ESBL)- or plasmid-mediated AmpC β-lactamase-producers by double disk synergy test, PCR and sequencing for β-lactamase genes. The most frequently identified ESBL gene among E. coli and K. pneumoniae isolates was bla SHV-12 and bla CTX-M ( bla CTX-M-9, bla CTX-M-14, bla CTX-M-3, and bla CTX-M-15). Four kinds of plasmid-mediated AmpC β-lactamases, ACT-1, CMY-1, CMY-2, and DHA-1, were detected. ESBL production was associated with high levels of resistance to tetracycline, sulfisoxazole, streptomycin, kanamycin, gentamicin and tobramycin when compared to non-ESBL producing isolates. Conclusively, this study suggests that the CTX-M β-lactamases are prevalent and various kinds of plasmid-mediated AmpC enzymes are distributed in clinical isolates of E. coli and Klebsiella spp. in Korea.

Antimicrobial susceptibility of clinical isolates of Enterobacteriaceae producing complex β-lactamase patterns including extended-spectrum enzymes

The antimicrobial susceptibility of 103 clinical isolates of Enterobacteriaceae to 11 antibiotics, was investigated, using a conventional inoculum size (5×10 5 CFU) and a higher inoculum size (5×10 8 CFU). All the isolates produced complex β-lactamase patterns, including an extended-spectrum β-lactamase (ESBL) of the TEM- or SHV-type plus other enzymes (a TEM-type or an SHV-type non-ESBL and/or a class C enzyme). The following repertoire of ESBLs was produced by the isolates: TEM-15, TEM-19, TEM-26, TEM-52, TEM-72, TEM-87, TEM-92, SHV-2a, SHV-5 and SHV-12, as assessed by sequencing. Production of the other enzymes was showed by analytical isoelectric focusing. Overall, meropenem was the most active agent and less influenced by inoculum size, while other β-lactams showed a lower activity and a significant inoculum size effect. In conclusion, from its in vitro performance, meropenem could be considered as the last resource drug against strains producing complex β-lactamase patterns including an ESBL.

Hydrolysis of third-generation cephalosporins by class C beta-lactamases. Structures of a transition state analog of cefotoxamine in wild-type and extended spectrum enzymes.

Bacterial resistance to the third-generation cephalosporins is an issue of great concern in current antibiotic therapeutics. An important source of this resistance is from production of extended-spectrum (ES) beta-lactamases by bacteria. The Enterobacter cloacae GC1 enzyme is an example of a class C ES beta-lactamase. Unlike wild-type (WT) forms, such as the E. cloacae P99 and Citrobacter freundii enzymes, the ES GC1 beta-lactamase is able to rapidly hydrolyze third-generation cephalosporins such as cefotaxime and ceftazidime. To understand the basis for this ES activity, m-nitrophenyl 2-(2-aminothiazol-4-yl)-2-[(Z)-methoxyimino]acetylaminomethyl phosphonate has been synthesized and characterized. This phosphonate was designed to generate a transition state analog for turnover of cefotaxime. The crystal structures of complexes of the phosphonate with both ES GC1 and WT C. freundii GN346 beta-lactamases have been determined to high resolution (1.4-1.5 Angstroms). The serine-bound analog of the tetrahedral transition state for deacylation exhibits a very different binding geometry in each enzyme. In the WT beta-lactamase the cefotaxime-like side chain is crowded against the Omega loop and must protrude from the binding site with its methyloxime branch exposed. In the ES enzyme, a mutated Omega loop adopts an alternate conformation allowing the side chain to be much more buried. During the binding and turnover of the cefotaxime substrate by this ES enzyme, it is proposed that ligand-protein contacts and intra-ligand contacts are considerably relieved relative to WT, facilitating positioning and activation of the hydrolytic water molecule. The ES beta-lactamase is thus able to efficiently inactivate third-generation cephalosporins.

VEB-1-like extended-spectrum beta-lactamases in Pseudomonas aeruginosa, Kuwait.

Two clinical Pseudomonas aeruginosa isolates from patients in intensive care units in Kuwait were resistant to expanded-spectrum cephalosporins and showed a synergistic effect between ceftazidime and clavulanic acid. This is the first report of extended-spectrum enzymes from nosocomial isolates from the Middle East.

Molecular and biochemical characterization of VEB-1, a novel class A extended-spectrum beta-lactamase encoded by an Escherichia coli integron gene.

A clinical isolate, Escherichia coli MG-1, isolated from a 4-month-old Vietnamese orphan child, produced a beta-lactamase conferring resistance to extended-spectrum cephalosporins and aztreonam. In a disk diffusion test, a typical synergistic effect between ceftazidime or aztreonam and clavulanic acid was observed along with an unusual synergy between cefoxitin and cefuroxime. The gene for VEB-1 (Vietnamese extended-spectrum beta-lactamase) was cloned and expressed in E. coli JM109. The recombinant plasmid pRLT1 produced a beta-lactamase with a pI of 5.35 and conferred high-level resistance to extended-spectrum (or oxyimino) cephalosporins and to aztreonam. Vmax values for extended-spectrum cephalosporins were uncommonly high, while the affinity of the enzyme for ceftazidime and aztreonam was relatively low. blaVEB-1 showed significant homology at the DNA level with only blaPER-1 and blaPER-2. Analysis of the deduced protein sequence showed that VEB-1 is a class A penicillinase having very low levels of homology with any other known beta-lactamases. The highest percentage of amino acid identity was 38% with PER-1 or PER-2, two uncommon class A extended-spectrum enzymes. Exploration of the genetic environment of blaVEB-1 revealed the presence of gene cassette features, i.e., (i) a 59-base element associated with blaVEB-1; (ii) a second 59-base element just upstream of blaVEB-1, likely belonging to the aacA1-orfG gene cassette; (iii) two core sites (GTTRRRY) on both sides of blaVEB-1; and (iv) a second antibiotic resistance gene 3' of blaVEB-1, aadB. blaVEB-1 may therefore be the first class A extended-spectrum beta-lactamase that is part of a gene cassette, which itself is likely to be located on a class 1 integron, as sulfamide resistance may indicate. Furthermore, blaVEB-1 is encoded on a large (> 100-kb) transferable plasmid found in a Klebsiella pneumoniae MG-2 isolated at the same time from the same patient, indicating a horizontal gene transfer.

Beta-Lactamase inhibition and in vitro activity of sulbactam and sulbactam/cefoperazone.

beta-Lactamase inhibitors such as sulbactam are beta-lactam compounds that have low antimicrobial activity but are able to inhibit enzymes (beta-lactamases) that destroy beta-lactam antibiotics like penicillins and cephalosporins. The main activity of beta-lactamase inhibitors is directed against plasmid-mediated transferable enzymes and various extended-spectrum enzymes. Sulbactam is also active against some of the fixed chromosomally mediated enzymes produced by gram-negative bacteria and is active against Bacteroides and Acinetobacter species. Cefoperazone, a third-generation cephalosporin, is active against a wide range of gram-positive and gram-negative bacteria, including Enterobacteriaceae and Pseudomonas species. However, transferable beta-lactamases are now produced by numerous gram-negative organisms, mitigating the effectiveness of therapy with this agent. Chromosomally mediated enzymes are less common but can be induced in some strains of Klebsiella, Enterobacter, and Serratia species. The full potential of cefoperazone against Enterobacteriaceae and Pseudomonas species is restored by the addition of sulbactam; this addition extends cefoperazone's spectrum of activity to include Bacteroides and Acinetobacter species.

Comparative activities of clavulanic acid, sulbactam, and tazobactam against clinically important beta-lactamases

Clavulanic acid, sulbactam, and tazobactam are inhibitors of a variety of plasmid-mediated beta-lactamases. However, inhibition data for these three inhibitors with a wide range of different plasmid-mediated beta-lactamases have not yet been compared under the same experimental conditions. A number of groups have inferred that clavulanic acid inhibits extended-spectrum TEM and SHV beta-lactamases, but inhibition data have rarely been published. In this study, the 50% inhibitory concentrations of these three beta-lactamase inhibitors for 35 plasmid-mediated beta-lactamases have been determined. Of these 35 beta-lactamases, 20 were extended-spectrum TEM- or SHV-derived beta-lactamases. The other 15 enzymes were conventional-spectrum beta-lactamases such as TEM-1 and SHV-1. Clavulanic acid was a more potent inhibitor than sulbactam for 32 of the 35 plasmid-mediated beta-lactamases tested. In particular, clavulanic acid was 60 and 580 times more potent than sulbactam against TEM-1 and SHV-1, respectively, currently the two most clinically prevalent gram-negative plasmid-mediated beta-lactamases. Statistical analysis of the data of the 50% inhibitory concentrations showed that clavulanic acid was 20 times more active overall than sulbactam against the conventional-spectrum enzymes. In addition, clavulanic acid was 14 times more potent than sulbactam at inhibiting the extended-spectrum enzymes. Tazobactam also showed significantly greater activity than sulbactam against the two groups of beta-lactamases. There were no significant differences between the overall activities of tazobactam and clavulanic acid against the extended-spectrum TEM and SHV enzymes and conventional-spectrum enzymes, although differences in their inhibition profiles were observed.

Importance of beta-lactamase stability in treating today's respiratory tract infections.

In respiratory tract infections Streptococcus pneumoniae, Moraxella catarrhalis, Haemophilus influenzae and Klebsiella spp. are the most frequently encountered bacterial pathogens. Resistance of clinical S. pneumoniae isolates is known to be independent of beta-lactamase production, whereas resistance of the other species mentioned is due to beta-lactamase production. With respect to M. catarrhalis the first beta-lactamase-producing (bla+) isolate was detected in clinical specimens in 1976 and now, 70-90% of all clinical isolates are bla+. The enzymes BRO-1 and BRO-2 are transposon-mediated thus explaining their rapid spread; they hydrolyze penicillin compounds very rapidly and, to a lesser extent, the older cephalosporins. Resistance in clinical H. influenzae isolates is mainly due to the prevalence of the most widespread transposon-mediated beta-lactamase, TEM-1, which has a substrate profile resembling that of the BRO enzymes. Bla+ H. influenzae isolates make up to 6% of the total in Northern Europe, in Southern Europe up to 55% and in many African countries more than 80%. More than 95% of Klebsiella spp. isolates possess a chromosomally encoded penicillinase and to a varying extent a plasmid-mediated enzyme in addition. Recently, reports from several countries have pointed to the clinical relevance of 'extended-spectrum enzymes' derived by point mutation from the 'classical' TEM-1 or TEM-2 enzymes. These new enzymes (TEM-3 to TEM-21) exhibit a broadened substrate profile, inactivating even the oxyiminocephalosporins. The most stable compounds are ceftibuten and cefetamet. With respect to the future, these enzymes may spread between species due to their location on transposons.(ABSTRACT TRUNCATED AT 250 WORDS)

Nucleotide sequence and phylogeny of SHV-2 beta-lactamase

We determined the nucleotide sequence of the blaSHV-2(pBP60-1) gene from Klebsiella ozaenae which confers resistance to broad-spectrum cephalosporins. The structural gene encodes a polypeptide product of 286 amino acids, and the estimated molecular weight of the mature protein is 28,900. Amino acid sequence comparison of the SHV-2pBP60-1 enzyme with all known class A beta-lactamases and homology studies showed that the residues were highly conserved. Furthermore, SHV-2pBP60-1 was clearly related to SHV-1, LEN-1, and OHIO-1. The SHV-2pBP60-1 enzyme differed from SHV-1 isolated from Klebsiella pneumoniae by seven amino acid substitutions. One of these substitutions, the Gly----Ser substitution at position 234, is probably a key region for the novel activity of cefotaxime hydrolysis. A phylogenetic tree was constructed by using all class A beta-lactamases of known sequences by a progressive alignment method. The data suggested that the beta-lactamases of gram-positive Streptomyces, Staphylococcus, and Bacillus species appeared early in evolution, followed by the PSE and CARB enzymes of Pseudomonas species and, more recently, by the SHV-type and TEM-type enzymes found in enteric bacteria. Larger evolutionary distances separated clusters of the gram-positive beta-lactamases than separated clusters of the gram-negative enzymes. Results of this phylogenetic study suggested that extended-spectrum enzymes are recent derivatives that are selected by the use of new cephalosporins.

Resistance to third generation cephalosporins: the current situation.

Newer beta-lactam antibiotics, notably the third generation cephalosporins (3 GC) have been designed for providing high intrinsic potency against a large variety of microorganisms. Bacterial resistance can occur however, and nowadays, clinicians are concerned by novel situations where even most recently developed compounds can be ineffective. A first situation is generated by bacteria which produce great amounts of chromosomal cephalosporinase. The resistance emerges during therapy, in hospital isolates which are classified as susceptible with conventional susceptibility testing. The prevalence of 3 GC resistance among these gram-negative rods with inducible beta-lactamase seems to increase in some institutions but the significance of susceptibility testing in this regard is doubtful. It is probably more important to note that the prevalence of gram-negative rods with inducible beta-lactamases remains stable. A second problem arose with the abrupt development of plasmid mediated beta-lactamases markedly active against 3 GC. This resistance is underestimated because some strains fall into susceptibility range of 3 GC as determined by MICs or inhibition zone sizes. These extended spectrum enzymes are now distributed over four continents and represent a growing threat.