C. Chanal

A Novel CTX-M β-Lactamase (CTX-M-8) in Cefotaxime-Resistant Enterobacteriaceae Isolated in Brazil

ABSTRACT To estimate the diversity of extended-spectrum β-lactamases in Brazil, 18 strains from different species of the familyEnterobacteriaceae exhibiting a positive double-disk synergy test were collected by a clinical laboratory from several hospitals in Rio de Janeiro, Brazil, in 1996 and 1997. Four strains (Proteus mirabilis, Enterobacter cloacae,Enterobacter aerogenes, and Citrobacter amalonaticus) hybridized with a 550-bp CTX-M probe. The P. mirabilis strain produced a CTX-M-2 enzyme. The E. cloacae, E. aerogenes, and C. amalonaticus isolates harbored a bla gene which was identified by cloning and sequencing as ablaCTX-M gene. E. coli HB101 transconjugants and the E. coli DH5α transformant harboring a recombinant plasmid produced a CTX-M β-lactamase with an isoelectric point of 7.6 conferring a resistance phenotype characterized by a higher level of resistance to cefotaxime than to ceftazidime, as observed with the other CTX-M enzymes. The deduced protein sequence showed a novel Ambler class A CTX-M enzyme, named CTX-M-8, which had 83 to 88% identity with the previously described CTX-M enzymes. The phylogenic study of the CTX-M family including CTX-M-8 revealed four CTX-M types, CTX-M-8 being the first member of a new phylum of CTX-M enzymes. The evolutionary distances between the four types of CTX-M were large, suggesting that the four clusters branched off early from a distant unknown enzyme and that intermediate enzymes probably existed.

Novel Cefotaximase (CTX-M-16) with Increased Catalytic Efficiency Due to Substitution Asp-240→Gly

ABSTRACT Three clinical strains (Escherichia coli Rio-6,E. coli Rio-7, and Enterobacter cloacae Rio-9) collected in 1996 and 1999 from hospitals in Rio de Janeiro (Brazil) were resistant to broad-spectrum cephalosporins and gave a positive double-disk synergy test. Two blaCTX-M genes encoding β-lactamases of pl 7.9 and 8.2 were implicated in this resistance: the blaCTX-M-9 gene observed inE. coli Rio-7 and E. cloacae Rio-9 and a novel CTX-M-encoding gene, designated blaCTX-M-16, observed in E. coli strain Rio-6. The deduced amino acid sequence of CTX-M-16 differed from CTX-M-9 only by the substitution Asp-240→Gly. The CTX-M-16-producing E. coli transformant exhibited the same level of resistance to cefotaxime (MIC, 16 μg/ml) but had a higher MIC of ceftazidime (MIC, 8 versus 1 μg/ml) than the CTX-M-9-producing transformant. Enzymatic studies revealed that CTX-M-16 had a 13-fold higher affinity for aztreonam and a 7.5-fold higher kcat for ceftazidime than CTX-M-9, thereby showing that the residue in position 240 can modulate the enzymatic properties of CTX-M enzymes. The two blaCTX-M-9 genes and the blaCTX-M-16 gene were located on different plasmids, suggesting the presence of mobile elements associated with CTX-M-encoding genes. CTX-M-2 and CTX-M-8 enzymes were found in Brazil in 1996, and two other CTX-M β-lactamases, CTX-M-9 and CTX-M-16, were subsequently observed. These reports are evidence of the diversity of CTX-M-type extended-spectrum β-lactamases in Brazil.

Molecular characterization of nine different types of mutants among 107 inhibitor-resistant TEM beta-lactamases from clinical isolates of Escherichia coli.

DNA-DNA hybridization and sequencing were performed to determine the molecular basis of resistance to clavulanic acid in 107 inhibitor-resistant TEM (IRT) enzymes produced by Escherichia coli clinical isolates. These beta-lactamases derived from TEM-1 enzyme focused at pI 5.2 (n = 68) or 5.4 (n = 39) and were very poorly inhibited by clavulanic acid compared with TEM-1 enzyme. Results showed that the amino acid sequences of 84 of the 107 enzymes differ from TEM-1 by one or two substitutions previously described: Arg-244-->Ser (IRT-2) in 22 strains, Met-69-->Leu (TEM-33) in 17 strains, Met-69-->Val (TEM-34) in 14 strains, Met-69-->Ile (IRT-3) in 6 strains, Met-69-->Leu associated with Asn-276-->Asp (IRT-4) in 13 strains, and Met-69-->Val associated with Asn-276-->Asp (TEM-36) in 12 strains. A new combination, Met-69-->Ile with Asn-276-->Asp, was found in 20 strains and was called IRT-8. Two IRT enzymes not previously described were characterized. The substitution Met-69-->Val associated with a novel substitution Arg-275-->Leu occurred in one strain. The combination Met-69-->Leu and Asn-276-->Asp was associated with the novel substitution Trp-165-->Arg in two strains. These two novel enzymes were called IRT-9 and IRT-10, respectively. The implication of these novel mutated positions, 165 and 275, in resistance to inactivation by clavulanate was supported by crystallographic data on the TEM-1 enzyme and results of site-directed mutagenesis. Molecular characterization of these mutants showed great diversity among the genes coding for inhibitor-resistant TEM enzymes produced by clinical E. coli isolates.

CTX-M-1, CTX-M-3, and CTX-M-14 β-Lactamases from Enterobacteriaceae Isolated in France

ABSTRACT Six clinical CTX-M-producing isolates of the family Enterobacteriaceae were detected between 1999 and 2000 in different French hospitals. Two strains produced CTX-M-1 and CTX-M-3 and four strains produced CTX-M-14, a mutant Ala-231→Val of CTX-M-9. A putative transposable element, ISEcp-1, was located 43 bp upstream of all the blaCTX-M genes. Two CTX-M-14-encoding plasmids exhibited similar restriction patterns. The CTX-M-1- and CTX-M-3-encoding plasmids were related to the CTX-M-1- and CTX-M-3-encoding plasmids previously reported in 1990 in France and in 1998 in Poland, respectively.

A 1998 Survey of Extended-Spectrum β-Lactamases in Enterobacteriaceae in France

ABSTRACT In a 3-month period in 1998, 79 consecutive isolates ofEnterobacteriaceae producing an extended-spectrum β-lactamase (ESBL) were collected. ESBLs were predominantly TEM derivatives (74 of 79): TEM-24-like (40 isolates), TEM-3-like (29 isolates), TEM-21 (3 isolates), and TEM-4 and TEM-52 (1 isolate each). Four isolates produced SHV derivatives SHV-4 (three isolates) and SHV-5 (one isolate), and one strain produced a CTX-M-3 enzyme. The high proportion of TEM-24-like-producing Enterobacter aerogenesisolates (36 of 79) suggests the occurrence of an epidemic strain in France.

A Novel Class A Extended-Spectrum β-Lactamase (BES-1) in Serratia marcescens Isolated in Brazil

ABSTRACT Serratia marcescens Rio-5, one of 18 extended-spectrum β-lactamase (ESBL)-producing strains isolated in several hospitals in Rio de Janeiro (Brazil) in 1996 and 1997, exhibited a high level of resistance to aztreonam (MIC, 512 μg/ml) and a distinctly higher level of resistance to cefotaxime (MIC, 64 μg/ml) than to ceftazidime (MIC, 8 μg/ml). The strain produced a plasmid-encoded ESBL with a pI of 7.5 whose bla gene was not related to those of other plasmid-mediated Ambler class A ESBLs. Cloning and sequencing revealed a bla gene encoding a novel class A β-lactamase in functional group 2be, designated BES-1 (Brazil extended-spectrum β-lactamase). This enzyme had 51% identity with chromosomal class A penicillinase of Yersinia enterocolitica Y56, which was the most closely related enzyme and 47 to 48% identity with CTX-M-type β-lactamases, which were the most closely related ESBLs. In common with CTX-M enzymes, BES-1 exhibited high cefotaxime-hydrolyzing activity (kcat, 425 s−1). However, BES-1 differed from CTX-M enzymes by its significant ceftazidime-hydrolyzing activity (kcat, 25 s−1), high affinity for aztreonam (Ki, 1 μM), and lower susceptibility to tazobactam (50% inhibitory concentration [IC50], 0.820 μM) than to clavulanate (IC50, 0.045 μM). Likewise, certain characteristic structural features of CTX-M enzymes, such as Phe-160, Ser-237, and Arg-276, were observed for BES-1, which, in addition, harbored different residues (Ala-104, Ser-171, Arg-220, Gly-240) and six additional residues at the end of the sequence. BES-1, therefore, may be an interesting model for further investigations of the structure-function relationships of class A ESBLs.

Chromosome-Encoded Class D β-Lactamase OXA-23 in Proteus mirabilis

ABSTRACT Ten nonrepetitive Proteus mirabilis isolates, which were collected over 4 years (1996 to 1999) at the teaching hospital of Clermont-Ferrand, France, produced class D carbapenemase OXA-23. MICs of imipenem were 0.25 to 0.5 μg/ml for these clinical isolates. Molecular typing revealed that the 10 P. mirabilis isolates originated from the same clonal strain. Hybridization of I-CeuI-generated chromosome fragments with a blaOXA-23 probe showed that the gene was chromosome encoded in the P. mirabilis strain.

Novel plasmid-mediated beta-lactamase in clinical isolates of Klebsiella pneumoniae more resistant to ceftazidime than to other broad-spectrum cephalosporins.

Multiresistant Klebsiella pneumoniae strains isolated from three patients in the same intensive care unit were more resistant to ceftazidime than to cefotaxime and aztreonam but remained susceptible to moxalactam and imipenem. Resistance to beta-lactams, kanamycin, streptomycin, sulfonamides, and tetracyclines was transferable to Escherichia coli by conjugation and was lost en bloc after treatment with ethidium bromide. Agarose gel electrophoresis of wild types and transconjugants indicated that these resistances were mediated by a 150-kilobase plasmid, pCFF14. The strains constitutively produced a beta-lactamase with isoelectric point close to 5.6 and which had a higher Vmax for ceftazidime and cephalothin than for cefotaxime. The substrate profile and isoelectric point of this enzyme thus differ from those of other known plasmid-mediated beta-lactamases, including the broad-spectrum enzyme CTX-1. Hybridization studies support the derivation of the novel enzyme from a TEM-type beta-lactamase. Images

First characterization of inhibitor-resistant TEM (IRT) beta-lactamases in Klebsiella pneumoniae strains.

Two clinical strains of Klebsiella pneumoniae, TP 01 and TP 02, presented resistance to amoxicillin-clavulanate and were fully susceptible to cephalothin. These strains produced two beta-lactamases, SHV-1 and a TEM enzyme with a pI of 5.2. The previously described changes Arg-244-->Cys and Arg-244-->Ser in IRT-1 and IRT-2 (A. Belaaouaj, C. Lapoumeroulie, M. M. Caniça, G. Vedel, P. Nevot, R. Krishnamoorthy, and G. Paul, FEMS Microbiol. Lett. 120:75-80, 1994) were found in TEM enzymes from the TP 01 and TP 02 strains, respectively. This is the first report of inhibitor-resistant TEM (IRT) in species other than Escherichia coli from the family Enterobacteriaceae.

Multiplicity of TEM-derived beta-lactamases from Klebsiella pneumoniae strains isolated at the same hospital and relationships between the responsible plasmids.

Five plasmid-mediated beta-lactamases conferring high-level resistance to ceftazidime were isolated from Klebsiella pneumoniae strains in the same hospital. These enzymes had isoelectric points ranging from 5.3 to 6.5 (CAZ-1, 5.55; CAZ-2, 6.0; CAZ-3, 5.3; CAZ-6, 6.5; and CAZ-7, 6.3). All isolates and their Escherichia coli transconjugants were highly resistant to amoxicillin (MICs, greater than 4,096 micrograms/ml), piperacillin (64 to 256 micrograms/ml), cephalothin (32 to 256 micrograms/ml), and ceftazidime (32 to 512 micrograms/ml) but remained moderately susceptible to cefotaxime (0.5 to 8 micrograms/ml). Only CAZ-6- and CAZ-7-producing strains were highly resistant to aztreonam (64 to 128 micrograms/ml). All the isolates remained susceptible to moxalactam and imipenem. The reduced activity of piperacillin, cefotaxime, ceftazidime, or aztreonam was restored by 2 micrograms of clavulanate, sulbactam, tazobactam, or brobactam per ml for E. coli producing CAZ-2, CAZ-3, and CAZ-7. Sulbactam had a lower protective effect than other inhibitors for E. coli harboring CAZ-1 and especially CAZ-6. Except for CAZ-1, which was mediated by a 150-kilobase (kb) plasmid (pCFF14), the other ceftazidimases were mediated by plasmids of 85 kb with EcoRI digestion patterns similar to that of pCFF04 encoding CTX-1 beta-lactamase. A TEM probe hybridized with a 19-kb EcoRI fragment of all these closely related plasmids. Images