Novel transferable extended-spectrum beta-lactamase (SHV-6) from Klebsiella pneumoniae conferring selective resistance to ceftazidime.
ABSTRACT A clinical isolate of Klebsiella pneumoniae sensu lato isolated from throat and a blood culture taken from a neutropenic patient treated for 2 weeks with ceftazidime and vancomycin was resistant to ceftazidime (MIC: 32 micrograms/ml) and moderately susceptible to aztreonam (MIC: 4 micrograms/ml). The isolate contained a plasmid of 180 kb which, when transferred to Escherichia coli by conjugation, conferred resistance to ceftazidime and tetracycline. The transconjugant had decreased susceptibility to ceftazidime (128-fold) and aztreonam (8-fold). Clavulanic acid and sulbactam each inhibited the resistance and clavulanic acid showed a synergistic effect when associated with ceftazidime and aztreonam. An extended-spectrum beta-lactamase with an isoelectric point of 7.6 was detected in the clinical isolates from blood and its transconjugant. This beta-lactamase showed similar substrate and inhibition profiles to SHV-1. In particular it did not hydrolyse ceftazidime. Hybridization with an intragenic probe for SHV-3 indicates that this beta-lactamase is an SHV-type enzyme. We propose that this novel CAZ-type extended-spectrum beta-lactamase be named SHV-6.
Article: Evolution of extended-spectrum beta-lactam resistance (SHV-8) in a strain of Escherichia coli during multiple episodes of bacteremia.[show abstract] [hide abstract]
ABSTRACT: Nine isolates of Escherichia coli were recovered from seven blood cultures over a period of 3 months from a 19-month-old female with aplastic anemia. Initial isolates were susceptible to extended-spectrum cephalosporins, including ceftazidime (MIC, < or = 0.25 microgram/ml), but gradually became resistant to this drug (MICs, > or = 128 micrograms/ml) and other cephalosporins and the monobactam aztreonam. Molecular typing methods, including plasmid profile analysis, pulsed-field gel electrophoresis, and arbitrarily primed PCR, indicated that the nine isolates were derived from a common ancestor. Dot blot hybridization and PCR analysis of total bacterial DNA using blaSHV- and blaTEM-specific DNA probes and primers identified the presence of a blaTEM beta-lactamase gene in all of the isolates and a blaSHV gene in the isolates with elevated ceftazidime MICs. Isoelectric focusing analysis of crude lysates showed that all nine isolates contained an enzyme with a pI of 5.4 corresponding to the TEM-1 beta-lactamase, and those isolates containing an SHV-type beta-lactamase demonstrated an additional band with a pI of 7.6. The first of the ceftazidime-resistant isolates appeared to hyperproduce the SHV enzyme compared to the other resistant isolates. DNA sequencing revealed a blaSHV-1 gene in the first ceftazidime-resistant isolate and a novel blaSHV gene, blaSHV-8, with an Asp-to-Asn substitution at amino acid position 179 in the remaining four isolates. Three of the ceftazidime-resistant isolates also showed a change in porin profile. The patient had received multiple courses of antimicrobial agents during her illness, including multiple courses of ceftazidime. This collection of blood isolates from the same patient appears to represent the in vivo evolution of resistance under selective pressure of treatment with various cephalosporins.Antimicrobial Agents and Chemotherapy 04/1997; 41(3):647-53. · 4.84 Impact Factor
Article: New system based on site-directed mutagenesis for highly accurate comparison of resistance levels conferred by SHV beta-lactamases.[show abstract] [hide abstract]
ABSTRACT: We developed a system based on site-directed mutagenesis that allows a precise comparison of SHV enzymes under isogenic conditions. In addition, the influences of two different, naturally occurring promoters were examined for each SHV derivative. The system comprised two separately cloned DNA fragments, each the size of 3.6 kb. Both fragments encoded an SHV gene originating from clinical isolates but with different promoters. The structural genes were made identical by site-directed mutagenesis. Other mutations were then introduced into both fragments by means of site-directed mutagenesis, resulting in the SHV derivatives SHV-1, SHV-2, SHV-2a, SHV-3, and SHV-5. The amino acid exchange of glutamic acid at position 235 for lysine in SHV-5 resulted in the highest resistance levels. SHV-3, differing from SHV-2 by the exchange of arginine at position 201 for leucine and previously described as indistinguishable from SHV-2, was shown to cause slightly higher resistance to ceftazidime and lower resistance to ceftriaxone, cefotaxime, and cefepime than SHV-2. The point mutation in SHV-2a, with the leucine-to-glutamine replacement at the unusual position 31, previously considered almost insignificant, proved to increase resistance to ceftazidime but reduced the MICs of all other cephalosporins tested when compared with those for SHV-2. For all clones harboring SHV derivatives, resistance was increased by a stronger promoter, in some cases masking the effect of the point mutation itself and demonstrating the importance of regulatory mechanisms of resistance.Antimicrobial Agents and Chemotherapy 09/1995; 39(8):1726-30. · 4.84 Impact Factor