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Antimicrob Agents Chemother. 2009 November; 53(11): 4955–4956.
Published online 2009 August 17. doi:  10.1128/AAC.00547-09
PMCID: PMC2772306

TEM-168, a Heretofore Laboratory-Derived TEM β-Lactamase Variant Found in an Escherichia coli Clinical Isolate[down-pointing small open triangle]

There have been over 160 variants of the TEM-1 and TEM-2 penicillinases described, most of which also exhibit activity against extended-spectrum cephalosporins (www.lahey.org/Studies/). It was noted in 1994 that a T265M mutation (Ambler numbering) was often associated with active-site mutations E104K, R164S, G238S, and E240K in some TEM variants (4). Comparison of MICs and kinetic studies of TEM-1 variants with the amino acid substitutions T265M, G238S, G238S:T265M, G238S:E240K, and G238S:G240K:T265M constructed by in vitro mutagenesis showed that the T265M mutation had no measurable effect on the TEM-1 wild type or on the G238S or G238S:240K variant. To date, no naturally occurring T265M variant of TEM-1 has been reported, though currently, 22 TEM variants contain an M265 residue along with other residue changes (www.lahey.org/Studies/).

Escherichia coli N08-1503 obtained by the National Microbiology Laboratory for advanced testing was resistant to ampicillin, cefazolin, and ampicillin-sulbactam, as determined by the submitting laboratory. Antimicrobial testing was carried out by disk diffusion and broth microdilution according to CLSI guidelines and Etests (AB Biodisk) according to manufacturer's instructions (Table (Table1)1) (1). The CLSI methods indicated that E. coli N08-1503 did not harbor an extended-spectrum β-lactamase (ESBL), though a reduced susceptibility to ceftazidime was noted. Analysis using the ESBL Etest strips yielded similar results. Interestingly, the cefepime ESBL Etest strip yielded a positive result. This strip is recommended outside of the United States when testing a strain with an inducible ampC gene (e.g., Enterobacter) or when a nondeterminable result is obtained. Broth microdilution confirmed the reduced susceptibility to cefepime and synergy with clavulanic acid, though the cefepime MICs were one or two doubling dilutions higher than the Etest results were. Resistance to amoxicillin-clavulanic acid and piperacillin-tazobactam and intermediate susceptibility to cefoxitin were also noted for E. coli N08-1503 and DH10B transformed with a plasmid (pT168) harboring blaTEM-168 (see below).

TABLE 1.
Antimicrobial data for E. coli N08-1503 and the transformants harboring pT168a

PCR analysis results for β-lactamase genes were positive for TEM and negative for SHV, CTX-M, OXA-1, and CMY-2 types. Sequence analysis of the TEM amplicon revealed a translation product with a single amino acid change from TEM-1, a T261M change (T265M by Ambler numbering) caused by a C782T transition. This variant has been assigned the name TEM-168 (www.lahey.org/Studies). In the blaTEM-168 promoter region, we detected the G162T change that defines the strong P4 promoter (3, 5). Plasmid analysis showed that an ~13-kb plasmid, pT168, harboring blaTEM-168 could be transformed into E. coli DH10B. Isoelectric focusing of crude extracts from E. coli N08-1503 and the DH10B transformant identified a single band of β-lactamase activity with a pI of 5.4. The resistance to β-lactam inhibitor combinations was likely due to the hyperproduction of plasmidic TEM-168 expressed from the P4 promoter, similar to what has been shown for TEM-1 (5, 6). Since the TEM-168 did not test positive with standard ESBL tests, it is easy to understand why this variant has not been described previously in clinical isolates. It was the unusual result with the cefepime ESBL Etest strip that made us continue our investigation to further characterize the TEM harbored by E. coli N08-1503.

Nucleotide sequence accession number.

The sequence of blaTEM-168 has been assigned accession number FJ919776 in the GenBank database.

Footnotes

[down-pointing small open triangle]Published ahead of print on 17 August 2009.

REFERENCES

1. Clinical and Laboratory Standards Institute. 2008. Performance standards for antimicrobial disk susceptibility tests. Document M02-A10. Clinical and Laboratory Standards Institute, Wayne, PA.
2. Clinical and Laboratory Standards Institute. 2009. Performance standards for antimicrobial susceptibility testing; 19th informational supplement. Document M100-S19. Clinical and Laboratory Standards Institute, Wayne, PA.
3. Goussard, S., and P. Courvalin. 1999. Updated sequence information for TEM β-lactamase genes. Antimicrob. Agents Chemother. 43:367-370. [PMC free article] [PubMed]
4. Huang, W., Q.-Q. Le, M. LaRocco, and T. Palzkill. 1994. Effect of threonine-to-methionine substitution at position 265 on structure and function of TEM-1 β-lactamase. Antimicrob. Agents Chemother. 38:2266-2269. [PMC free article] [PubMed]
5. Lartigue, M. L., V. Leflon-Guibout, L. Poirel, P. Nordmann, and M.-H. Nicolas-Chanoine. 2002. Promoters P3, Pa/Pb, P4, and P5 upstream from blaTEM genes and their relationship to β-lactam resistance. Antimicrob. Agents Chemother. 46:4035-4037. [PMC free article] [PubMed]
6. Wu, P. J., K. Shannon, and I. Phillips. 1994. Effect of hyperproduction of TEM-1 β-lactamase on in vitro susceptibility of Escherichia coli to β-lactam antibiotics. Antimicrob. Agents Chemother. 38:494-498. [PMC free article] [PubMed]

Articles from Antimicrobial Agents and Chemotherapy are provided here courtesy of American Society for Microbiology (ASM)