The antibiotic Nitrofurantoin is indicated for the treatment of acute or recurrent lower urinary tract infections (UTI) which is caused by strains of Escherichia coli, enterococci, staphylococci, Citrobacter, Klebsiella or Enterobacter. This study analysed ESBL-producing bacteria isolated from 300 urine samples of UTI patients from three referral hospitals in North Wales, UK. Multiplex PCR amplification of β-lactamase genes of the blaCTX-M groups 1, 2, 9 and 8/25, followed by DNA sequencing revealed one new beta-lactamase gene which is closely related to CTXM‑14 at the protein levels. Recombinant expression of this new bla gene renders Escherichia coli highly resistant to Nitrofurantoin. To characterize this new beta-lactamase, the enzyme was purified from the periplasmatic, space of E.coli or from the growth medium in a highly pure form utilising a C-terminal strep-tag. Isolectric focusing and phostag electrophoresis revealed a high degree of phosphorylation and the presence of at least two distinct protein species. Since this new gene was isolated only from one of the three hospitals, it is quite possible that the new beta-lactamase is responsible for a local increase in Nitrofurantoin resistance in UTI patients.