Evolution of extended-spectrum beta-lactam resistance (SHV-8) in a strain of Escherichia coli during multiple episodes of bacteremia

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.

Mechanisms of Antimicrobial Resistance in Bacteria

“…(The change in a single amino acid, from aspartate to asparagine, at position 178, as documented by DNA sequence analysis of the resistance gene, was responsible for the increased resistance level). 30 Polymerase chain reaction assays, designed to detect various ␤-lactamase resistance genes, confirmed that the gene encoding the TEM-1 ␤-lactamase was present in all of the isolates, including those that were initially susceptible to third-generation cephalosporins, and indicated the presence of a second ␤-lactamase gene (the SHV type) in isolates that were resistant to third-generation cephalosporins (data not shown).…”

“…The ESBLs are not active against cephamycins, such as cefoxitin and cefotetan [27][28][29][30] ; however, resistance to cephamycins and other ␤-lactams may arise as a result of changes in the porins in the outer membrane (proteins that form the water-filled channels through which drugs and other molecules enter the bacterial cell). Such changes decrease or eliminate the flow of small hydrophilic molecules like ␤-lactam drugs across the membrane.…”

What’s new in β-lactamases?

“…In a recent survey of detection of ESBLs in clinical isolates, Tenover and colleagues [37] found that only 18% of laboratories both correctly identified challenge organisms as potential ESBL producers and reported the susceptibility of oxyimino-cephalosporins to be resistant. Furthermore, false-positive results can be obtained with strains expressing a high level of a non-ESBL ␤-lactamase, such as SHV-1 [38,39].…”

Identification of bla KPC-2 on different plasmids of three Morganella morganii isolates

“…The special primers and amplification conditions were in accordance with references [10][11][12][13][14][15][16][17][18][19]. The positive PCR products were screened by electrophoresis at 1.5% agarose gel and were then sequenced by Shanghai Majorbio BioPharm Technology Co., Ltd., China.…”