Structural features related to hydrolytic activity against ceftazidime of plasmid-mediated SHV-type CAZ-5 beta-lactamase

Abstract

Tryptic peptides of the novel ceftazidimase CAZ-5 were sequenced by manual Edman degradation and aligned according to strong homology (more than 98%) with SHV-1 and SHV-2 13-lactamase sequences. differed from SHV-1 by five amino acid substitutions. Unusually high activity of CAZ-5 towards ceftazidime was imputed to substitution of a Lys for a Glu at position 214 of the mature protein.Broad-spectrum cephalosporins such as cefotaxime and related compounds, which are very effective against gramnegative bacteria, have been extensively used in hospital practice. However, infections caused by members of the family Enterobacteriaceae resistant to newer cephalosporins were soon observed (23,24). In part, the resistance was related to overproduction of chromosomally mediated cephalosporinase involving a stable derepression of enzyme synthesis (17,20). Otherwise, transferable resistance to broad-spectrum cephalosporins, first detected in the Federal Republic of Germany (15, 16), was responsible, especially in France, for outbreaks of nosocomial infections (6,14,26). Whatever the main bacterial phenotype (cefotaxime resistance, ceftazidime resistance, or multiresistance), it was associated with the synthesis of new plasmid-mediatedlactamases (5,7,8,10,12,21,25).Recently, such as enzyme, CAZ-5, also called SHV-4 (22), was isolated (Hopital Begin, St. Mandd, France) from Klebsiella pneumoniae 210-2, a strain resistant to broad-spectrum cephalosporins. The kinetic constants of CAZ-5 did not notably differ from those of the cefotaxime-hydrolyzing ,B-lactamase SHV-2, with the exception of the hydrolysis rates for ceftazidime and aztreonam, which were markedly increased ( markedly from that of the SHV-1 protein (3) and was in good agreement with that deduced from the amino acid sequence analysis. Eight first N-terminal sequence analyses each yielded one residue with the sequence Ser-Pro-Gln-ProLeu-Glu-Gln-Ile-identical to those of SHV-1 and SHV-2.The S-carboxymethylated protein was digested by trypsin (Sigma Chemical Co.), and the resulting peptide mixture was resolved by high-performance liquid chromatography on a Waters ,uBondapak C18 column (10 pum; 4 by 300 mm) (Fig. 1). Elution was performed with 0.1% trifluoroacetic acid in water as solvent A and acetonitrile as solvent B. A threestep linear gradient of solvent B was used (0 min, 5%; 35 min, 30%; 45 min, 45%; 60 min, 80%) with a flow rate of 1 ml/min. Apart from peak 16, which exhibited a higher hydrophobicity, the elution pattern of the tryptic peptides of CAZ-5 was identical to that of SHV-2 (2). Peaks 11 to 14, 3, and 5 to 22 were further chromatographed at pH 6.0 on the same column, with a linear gradient from 0.25 mM ammonium acetate buffer to 0.5 mM ammonium acetate bufferacetonitrile (2:3, vol/vol). Peptide T-19, which eluted with

Extended-Spectrum β-Lactamase-Producing Enterobacteriaceae in Community and Private Health Care Centers

“…Two strains of K. pneumoniae contained TEM-15, another one synthesized TEM-19, and the remaining strain of E. aerogenes produced the TEM-3 ESBL (Table 3). The single SHV-type ESBL was an SHV-4 enzyme produced by a K. pneumoniae strain (40). Likewise, one strain of E. coli that appeared to be highly resistant to cefotaxime but susceptible to ceftazidime produced a CTX-M-1 ␤-lactamase (8).…”

Characterization of the Extended-Spectrum β-Lactamase Reference Strain, Klebsiella pneumoniae K6 (ATCC 700603), Which Produces the Novel Enzyme SHV-18

“…Substitution of lysine for glutamate at position 240 of SHV-18, also seen in a number of other variants, including SHV-4 (39), SHV-5 (4), SHV-7 (8), and SHV-12 (35), is thought to have little effect on the hydrolysis of cefotaxime (16) but is necessary for resistance to ceftazidime and aztreonam (4,16,39). A high-level of resistance to ceftazidime, however, is achieved only by strains producing an SHV enzyme containing both serine at position 238 and lysine at position 240 (16).…”

A Review of SHV Extended-Spectrum β-Lactamases: Neglected Yet Ubiquitous

“…Only antibiotics for which 3 or more SHV enzyme values were available are reported . NA, not able to determine the rate of hydrolysis and affinity; NH, not hydrolyzed; ND, not determined .** Non ESBL SHV-1 is provided as reference .$ K cat /K m values are expressed as mM/s .# K cat /K m values are expressed as μM/s .* Values (Except IC 50 ) represent mean ± standard deviation . References: SHV-1 (Gutmann et al, 1989; Poirel et al, 2003); SHV-2 (Gutmann et al, 1989; Bradford et al, 1995; Winkler and Bonomo, 2016); SHV-2a (Podbielski et al, 1991); SHV-4 (Péduzzi et al, 1989); SHV-5 (Gutmann et al, 1989); SHV-7 (Bradford et al, 1995); SHV-9 (Prinarakis et al, 1996); SHV-13 (Yuan et al, 2000); SHV-18 (Rasheed et al, 2000); SHV-24 (Kurokawa et al, 2000); SHV-38 (Poirel et al, 2003); SHV-55 (Mendonça et al, 2006); SHV-57 (Ma et al, 2005): SHV-99 (Ramdani-Bouguessa et al, 2011); SHV-104 (Ben Achour et al, 2014); SHV-129 (Winkler and Bonomo, 2016) .…”