Identification of a CTX-M-255 ß-lactamase containing a G239S substitution selectively conferring resistance to penicillin/ß-lactamase inhibitor combinations.

OBJECTIVES: An Escherichia coli isolate, WGS1363, showed resistance to piperacillin/tazobactam but susceptibility to cephalosporins and contained a previously unrecognized ß-lactamase, CTX-M-255, as the only acquired ß-lactamase. CTX-M-255 was identical to CTX-M-27 except for a G239S substitution. Here, we characterize the hydrolytic spectrum of CTX-M-255 and a previously reported ß-lactamase, CTX-M-178, also containing a G239S substitution and compare it to their respective parental enzymes, CTX-M-27 and CTX-M-15. METHODS: All ß-lactamase genes were expressed in E. coli TOP10 and MICs to representative ß-lactam-antibiotics were determined. Furthermore, blaCTX-M-15,  blaCTX-M-27, blaCTX-M-178 and blaCTX-M-255 with C-terminal His-tag fusions were affinity purified for enzyme kinetic assays determining Michaelis-Menten kinetic parameters against representative ß-lactam-antibiotics and IC50s of clavulanate, sulbactam, tazobactam and avibactam. RESULTS: TOP10-transformants expressing blaCTX-M-178 and blaCTX-M-255 showed resistance to penicillin/ß-lactamase combinations and susceptibility to cephalothin and cefotaxime in contrast to transformants expressing blaCTX-M-15 and blaCTX-M-27. Determination of enzyme kinetic parameters showed that CTX-M-178 and CTX-M-255 both lacked hydrolytic activity against cephalosporins and showed impaired hydrolytic efficiency against penicillin antibiotics compared to their parental enzymes. Both enzymes appeared more active against piperacillin compared to benzylpenicillin and ampicillin. Compared to their parental enzymes, IC50s of ß-lactamase-inhibitors were increased more than 1000-fold for CTX-M-178 and CTX-M-255. CONCLUSIONS: CTX-M-178 and CTX-M-255, both containing a G239S substitution, conferred resistance to piperacillin/tazobactam and may be characterized as inhibitor-resistant CTX-M ß-lactamases. Inhibitor resistance was accompanied by loss of activity against cephalosporins and monobactams. These findings add to the necessary knowledge base for predicting antibiotic susceptibility from genotypic data.

Efficacy of piperacillin-tazobactam and cefotaxime against Escherichia coli hyperproducing TEM-1 in a mouse peritonitis infection model.

OBJECTIVES: Piperacillin-tazobactam (TZP) is a frequently prescribed antibiotic in hospital settings. Reports suggest in vivo efficacy of TZP, despite in vitro resistance of isolates susceptible to cephalosporins. Escherichia coli (E. coli) isolates hyperproducing TEM-1 ß-lactamase possess this phenotype. This study investigated the influence of tazobactam (TAZ) concentration on piperacillin (PIP) inhibition of such isolates and compared the in vivo efficacy of TZP with cefotaxime (CTX) in an infection model. METHODS: The PIP MICs for E. coli isolates, either hyperproducing TEM-1 because of promoter substitutions (n = 4) or because of gene amplification (n = 2) or producing an inhibitor-resistant TEM-35 (IRT) (n = 1), were determined using increasing concentrations of TAZ in a checkerboard setup. Furthermore, the efficacy of TZP and CTX against the isolates was investigated in a mouse peritonitis model using antibiotic exposures mimicking human conditions. Isolates producing either OXA-48 or CTX-M-15 ß-lactamases were included as controls. RESULTS: Using TAZ concentrations ≤ 64 mg/L, one isolate hyperproducing TEM-1 had a PIP MIC of 8 at TAZ 16 mg/L and two additional isolates at TAZ 64 mg/L. In the mouse peritonitis infection model, reduction of bacterial load in the peritoneum was larger for TZP than CTX only for the CTX-M-15-producing isolate. Larger reductions in bacterial load were observed after CTX treatment than TZP treatment for seven of the eight remaining test isolates. CONCLUSIONS: Piperacillin-tazobactam treatment of E. coli isolates hyperproducing TEM-1 was less effective than CTX treatment and may, for some isolates, be comparable with TZP treatment of isolates producing established resistance markers as IRT or OXA-48.

Resistance to piperacillin/tazobactam in Escherichia coli resulting from extensive IS26-associated gene amplification of blaTEM-1.

BACKGROUND: bla TEM-1 encodes a narrow-spectrum ß-lactamase that is inhibited by ß-lactamase inhibitors and commonly present in Escherichia coli. Hyperproduction of blaTEM-1 may cause resistance to penicillin/ß-lactamase inhibitor (P/BLI) combinations. OBJECTIVES: To characterize EC78, an E. coli bloodstream isolate, resistant to P/BLI combinations, which contains extensive amplification of blaTEM-1 within the chromosome. METHODS: EC78 was sequenced using Illumina and Oxford Nanopore Technology (ONT) methodology. Configuration of blaTEM-1 amplification was probed using PCR. Expression of blaTEM-1 mRNA was determined using quantitative PCR and ß-lactamase activity was determined spectrophotometrically in a nitrocefin conversion assay. Growth rate was assessed to determine fitness and stability of the gene amplification was assessed by passage in the absence of antibiotics. RESULTS: Illumina sequencing of EC78 identified blaTEM-1B as the only acquired ß-lactamase preceded by the WT P3 promoter and present at a copy number of 182.6 with blaTEM-1B bracketed by IS26 elements. The chromosomal location of the IS26-blaTEM-1B amplification was confirmed by ONT sequencing. Hyperproduction of blaTEM-1 was confirmed by increased transcription of blaTEM-1 and ß-lactamase activity and associated with a significant fitness cost; however, the array was maintained at a relatively high copy number for 150 generations. PCR screening for blaTEM amplification of isolates resistant to P/BLI combinations identified an additional strain containing an IS26-associated amplification of a blaTEM gene. CONCLUSIONS: IS26-associated amplification of blaTEM can cause resistance to P/BLI combinations. This adaptive mechanism of resistance may be overlooked if simple methods of genotypic prediction (e.g. gene presence/absence) are used to predict antimicrobial susceptibility from sequencing data.