SHV-16, a beta-lactamase with a pentapeptide duplication in the omega loop.

A clinical isolate of Klebsiella pneumoniae was found to be resistant to ampicillin (MIC of 128 microg/ml), ticarcillin (MIC of 512 microg/ml), and ceftazidime (MIC of 128 microg/ml) and susceptible to all other beta-lactams; a synergistic effect between clavulanate and ceftazidime suggested the presence of an extended-spectrum beta-lactamase (ESBL). Transconjugants in Escherichia coli were obtained at low levels (10(-7) per donor cell) and exhibited a similar beta-lactam resistance pattern (resistant to ampicillin, ticarcillin, and ceftazidime at 64 microg/ml). The ESBL, pI 7.6, was encoded by a large plasmid (>100 kb) which did not carry any other resistance determinant. The ESBL-encoding gene was amplified by PCR using bla(SHV)-specific primers and was sequenced. The deduced amino acid sequence of the SHV-16 ESBL showed that it differed from SHV-1 by only a pentapeptide insertion (163DRWET167) corresponding to a tandem duplication in the omega loop. The implication of the 163a-DRWET163b-DRWET sequence in ceftazidime resistance was confirmed by cloning either bla(SHV-1) or bla(SHV-16) in the same vector, subsequently introduced in the same E. coli strain. Under these isogenic conditions, SHV-16 conferred a 32-fold increase in ceftazidime MIC compared to that with SHV-1. Furthermore, site-directed mutagenesis experiments modifying either E166aA or E166bA revealed that the functional glutamic residue was that located in the first copy of the duplicated sequence. But surprisingly, the second E166b also conferred a low-level resistance to ceftazidime. This work is the first description of a class A enzyme exhibiting an extended substrate specificity due to an insertion instead of a nucleotide substitution(s) in a clinical isolate.

Molecular epidemiology of an outbreak due to extended-spectrum beta-lactamase-producing enterobacteria in a French hospital.

Between June 1992 and June 1993, 128 extended-spectrum beta-lactamase-producing enterobacteria (123 Klebsiella pneumoniae, 3 Escherichia coli, 1 Enterobacter aerogenes, and 1 Citrobacter diversus) were collected in a French university hospital. These isolates were recovered mainly from patients hospitalized in intensive care and neurosurgery units. The 128 strains were divided into 14 antibiotypes (ATBs; ATB1 to ATB14); 102 of 103 nonredundant isolates were shown to produce an SHV-4-related extended-spectrum beta-lactamase (pl 7.8, hybridization with a blaSHV probe); the remaining strain (Kp 2108) produced a TEM-3-related extended-spectrum beta-lactamase (pl 6.3, hybridization with a blaTEM probe). For representative isolates, five plasmid profiles (Pl to Pll-4), eight ribotypes (E1 to E8), and seven arbitrarily primed polymerase chain reaction profiles (A1 to A7) were obtained. The results suggest the spread of an epidemic strain of Klebsiella pneumoniae (E1, A1, Pl, various ATBs) from an intensive care unit throughout the hospital. Another epidemic strain (E2, A2, pl, ATB4) was confined to the neurosurgery unit. Other extended-spectrum beta-lactamase-producing Klebsiella pneumoniae of ribotypes distinct from E1 or E2 might result from the spread of an epidemic plasmid, such as reported for isolates of other enterobacterial species. Conversely, they might represent imported cases, such as the strain Kp 2108, which produced a TEM-3-related beta-lactamase.

[Comparative in vitro activity of cefepime: multicenter study in Aquitaine]. / Activité in vitro comparée du céfépime: étude multicentrique en Aquitaine.

Over a 6 month-period (1st January to 30th June 1995), the results of antibiotic susceptibility testing routinely performed for beta-lactams against enterobacteria, Pseudomonas aeruginosa, and Acinetobacter in 7 laboratory hospitals of Aquitaine, have been collected and divided in susceptibility profiles. A total of 9269 strains (7323 enterobacteria, 1667 P. aeruginosa, 279 Acinetobacter) have been examined. On the whole, cefepime (91,5%) and ceftazidime (91,7%) were the most active cephalosporins, followed by cefpirome (87,9%) and cefotaxime (80,4%); imipenem was the most active beta-lactam agent (97,4%). When the strains were divided according to their susceptibility profiles, the advantage of cefepime was shown to be related to its excellent activity against enterobacteria: all strains susceptible to cefotaxime and ceftazidime (CTX/CAZ-S) were susceptible to cefepime, as were most of the strains with an intermediate susceptibility or resistant to these drugs (CTX/CAZ-I/R, approximately 5% of the enterobacteria). The latter strains exhibited a phenotype corresponding either to the overproduction of their chromosomal cephalosporinase (approximately 20% of the species belonging to group 3) or to the synthesis of an extended spectrum beta-lactamase (19% of the strains of Klebsiella pneumoniae). Cefepime was active against 93% of the derepressed mutants of enterobacteria, including 3 imipenem resistant isolates of Enterobacter. CAZ-S strains of P. aeruginosa (84%) were usually susceptible to cefepime (80%), as were 6% of the CAZ-I/R strains. CAZ-S strains of A. baumannii (16.3%) were generally susceptible to cefepime (83%), as were 3.2% of the CAZ-I/R strains.