No abstract

Kluyvera ascorbata produces a ␤-lactamase that results in an atypical susceptibility pattern, including lowlevel resistance to penicillins, cephalothin, and cefuroxime, but this resistance is reversed by clavulanate. Ten nucleotide sequences of the corresponding gene, bla KLUA , were obtained and were found to have minor variations (96 to 100%). Otherwise, bla KLUA was found to be similar (95 to 100%) to some plasmid-encoded CTX-M-type ␤-lactamases. Finally, mobilization of bla KLUA on a plasmid was found to be mediated probably by a genetic mobile element like ISEcp1.

Beta-lactamases represent the main mechanism of bacterial resistance to beta-lactam antibiotics. The recent emergence of bacterial strains producing inhibitor-resistant TEM (IRT) enzymes could be related to the frequent use of beta-lactamase inhibitors such as clavulanic acid, sulbactam and tazobactam in hospitals and in general practice. The IRT beta-lactamases differ from the parental enzymes TEM-1 or TEM-2 by one, two or three amino acid substitutions at different locations. This paper reviews the phenotypic, genetic and biochemical characteristics of IRT beta-lactamases in an attempt to shed light on the pressures that have contributed to their emergence.

Approximately 10% (89 isolates) of Klebsiella pneumoniae isolated in 1985 from patients in intensive care units in Clermont-Ferrand exhibited a complex resistance phenotype towards antibiotics. They were resistant to amino-, carboxy- and ureidopenicillins, aminoglycosides (except gentamicin), chloramphenicol, sulphonamides, tetracyclines and, most importantly, to cephalosporins (except cefoxitin and latamoxef) and to aztreonam. The metabolic profile of fifty isolates was identical and seven were selected for further study. All the resistance characters in these isolates were transferable to Escherichia coli by conjugation and were lost en bloc after treatment with ethidium bromide. Agarose gel electrophoresis of crude lysates of the wild types and their transconjugants indicated that the multiple resistances were mediated by a 95kb plasmid, pCF04. The seven isolates selected for study and their corresponding transconjugants, constitutively produced a plasmid-mediated beta-lactamase with a pI of 6.3 that was much more active against third-generation cephalosporins than against cephalothin. The substrate profile and the isoelectric-focusing behaviour of this enzyme differed from those of other known plasmid-mediated beta-lactamases, and the enzyme was designated CTX-1. A chromosomally-encoded SHV-1 (PIT-2) penicillinase (pI 7.7) was also present in the seven K. pneumoniae isolates but did not transfer. Resistance to aminoglycosides in the K. pneumoniae isolates was due to synthesis of a 6'-aminoglycoside acetyltransferase type IV. Our data indicate an epidemic of antibiotic multiply-resistant strains of K. pneumoniae producing a new beta-lactamase.

A clinical isolate ofPseudomonas aeruginosa RNL-1 showed resistance to extended-spectrum cephalosporins which was inhibited by clavulanic acid. Although this strain contained three plasmids ca. 80, 20, and 4 kb long, the resistance could not be transferred by mating-out assays with P. aeruginosa or Escherichia coli. Cloning of a 2.1-kb Sau3A fragment from P. aeruginosa RNL-1 into plasmid pACYC184 produced pPZ1, a recombinant plasmid that encodes a 1-lactamase. This f-lactamase (PER-1) had a relative molecular mass of 29 kDa and a pl of 5.4 and was biosynthesized by P. aeruginosa RNL-1 along with a likely cephalosporinase with a pl of 8.7. PER-1 showed a broad substrate profile by hydrolyzing benzylpenicillin, amoxicillin, ticarcillin cephalothin, cefoperazone, cefuroxime, HR 221, ceftriaxone, ceftazidime, and (moderately) aztreonam but not oxacillin, imipenem, or cephamycins. Vmax values for extended-spectrum cephalosporins were uncommonly high, and the affinity of the enzyme for most compounds was relatively low (i.e., high Km)* PER-1 activity was inhibited by clavulanic acid, sulbactam, imipenem, and cephamycins but not by EDTA. A 1.1-kb SnaBI fragment from pPZ1 failed to hybridize with plasmids that encode TEM-, SHV-, OXA-, or CARB/PSE-type 13-lactamase or with the ampC gene of P. aeruginosa. However, the same probe appeared to hybridize with chromosomal but not plasmid DNA from P. aeruginosa RNL-1. This study reports the properties of a novel extended-spectrum 13-lactamase in P. aeruginosa which may not be derived by point mutations from previously known enzymes of this species.More than 50 biochemically distinct P-lactamases responsible for resistance to ,-lactams have been reported in gram-negative bacteria. The resistance of broad-spectrum cephalosporins to these ,B-lactamases was a widely accepted concept in the beginning of the 1980s. However, overproduction of chromosomally mediated cephalosporinases has been described as responsible for failure of treatment of gram-negative bacterial infections with extended-spectrum cephalosporins (39). Since 1983, plasmid-mediated extended-spectrum 3-lactamases have been reported, primarily in Kiebsiella pneumoniae and then in numerous Enterobacteraceae species (16, 34). These enzymes hydrolyze extended-spectrum cephalosporins and aztreonam to various extents but usually neither cephamycins (cefoxitin and moxalactam) nor carbapenems (imipenem and meropenem). A common feature of these enzymes is inhibition of their activity by clavulanic acid. These enzymes are Ambler class A 1-lactamases, members of the TEM or SHV series that differ by a few point mutations in their structural genes (16,34 resistance to extended-spectrum cephalosporins. In this species, TEM-1 and TEM-2 1-lactamases confer additional resistance to ureidopenicillins (26). The OXA-type (oxacillin-hydrolyzing) enzymes possess high-level hydrolytic activity against cloxacillin, oxacillin, and methicillin (9, 10). Their activities are inhibited by clavulanic acid but to a lesser extent than TEM-or SHV-der...

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 bla CTX-M genes encoding ␤-lactamases of pl 7.9 and 8.2 were implicated in this resistance: the bla CTX-M-9 gene observed in E. coli Rio-7 and E. cloacae Rio-9 and a novel CTX-Mencoding gene, designated bla CTX

A clinical isolate of Enterobacter cloacae, strain NOR-1, exhibited resistance to imipenem and remained susceptible to extended-spectrum cephalosporins. Clavulanic acid partially restored the susceptibility of the strain to imipenem. Two 13-lactamases with isoelectric points (pI) of 6.9 and >9.2 were detected in strain E.cloacae NOR-1; the higher pl corresponded to AmpC cephalosporinase. Plasmid DNA was not detected in E. cloacae NOR-1 and imipenem resistance could not be transferred into Escherichia coli JM109. The carbapenem-hydrolyzing 13-lactamase gene was cloned into plasmid pACYC184. One recombinant plasmid, pPTN1, harbored a 5.3-kb Sau3A fragment from E. cloacae NOR-1 expressing the carbapenem-hydrolyzing 13-lactamase. This enzyme (pl 6.9) hydrolyzed ampicillin, cephalothin, and imipenem more rapidly than it did meropenem and aztreonam, but it hydrolyzed extended-spectrum cephalosporins only weakly and did not hydrolyze cefoxitin. Hydrolytic activity was partially inhibited by clavulanic acid, sulbactam, and tazobactam, was nonsusceptible to chelating agents such as EDTA and 1,10-o-phenanthroline, and was independent of the presence of ZnCl2. Its relative molecular mass was 30,000 Da. Induction experiments concluded that the carbapenem-hydrolyzing ,3-lactamase biosynthesis was inducible by cefoxitin and imipenem. Subcloning experiments with HindIII partial digests of pPTN1 resulted in a recombinant plasmid, designated pPTN2, which contained a 1.3-kb insert from pPTN1 and which conferred resistance to 13-lactam antibiotics.Hybridization studies performed with a 1.2-kb Hindlll fragment from pPTN2 failed to determine any homology with ampC of E. cloacae, with other known 13-lactamase genes commonly found in members of the family Enterobacteriaceae (blaTEM-l and blasHv.3 derivatives), and with previously described carbapenemase genes such as those from Xanthomonas maltophilia, Bacilus cereus, Bacteroidesfragilis (cfA), and Aeromonas hydrophila (cphA). This work describing the biochemical properties of a novel chromosome-encoded 13-lactamase from E. cloacae indicates that this enzyme differs from all the previously described carbapenemases. This is the first reported cloning of a Enterobacteriaceae.

To estimate the diversity of extended-spectrum ␤-lactamases in Brazil, 18 strains from different species of the family Enterobacteriaceae 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 a bla CTX-M gene. E. coli HB101 transconjugants and the E. coli DH5␣ transformant harboring a recombinant plasmid produced a CTX-M ␤-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.