Antibiotic resistance is considered a major public health concerns in the 21st century. Microbes that were once susceptible to antibiotics are becoming more and more difficult to treat as they acquire rapidly a wide range of resistance mechanisms including enzymes of the β-lactamase family. These proteins are typically plasmid-borne and inactivate beta-lactam ring containing antibiotics that are worldwide used to treat infections. This enzyme family with more than 2800 proteins is rapidly evolving through the spontaneous acquisition of missense mutations that enhance protein stability, increase catalytic turn-over and/or improve substrate specificity. This study is an epidemiological snapshot of the antibiotic resistance conferred by CTX-M-type extended-spectrum β-lactamases (CTX-M type ESBL) in patients based in North Wales who were diagnosed with urinary tract infection (UTI). Multiplex PCR amplification of β-lactamase genes of the blaCTX-M groups 1, 2, 9, and 8/25 followed by DNA sequencing of ESBL-producing isolates was performed with samples from three referral hospital in North Wales; Ysbyty Gwynedd, Glan Clwyd Hospital, and Wrexham Maelor Hospital In line with previous studies, the β-lactamase CTX-M-15 was found to be the dominant CTX-M variant in the study region. The analysis of samples from Ysbyty Gwynedd hospital at Bangor revealed however an as yet unknown CTX-M variant that differed from CTX-M-14 by only three missense mutations. The amino acid substitutions A55T, A273P and R277C reside in the beta-strand domain opposite of the active site of the enzyme. CTX-M-15, CTX-M-14 and the novel variant were expressed in the periplasm of recombinant E.coli using the inducible vector pASK-IBA2C. Determination of the Minimum Inhibitory Concentration (MIC) of a series of clinical relevant antibiotics using the induced recombinant strains showed a significant increase in Nitrofurantoin resistance and a partial increase in Cefoxitin resistance that were both specifically linked with the three mutations. In vitro Kinetic studies using protein from periplasmic extracts of induced strains showed however that neither the affinity for Nitrofurantoin nor for Cefoxitin was increased by these substitutions. Taken together, the three missense mutations in CTX-M-14 may render UTI patients isolates more resistant to Nitrofurantoin because the three amino acid changes increase enzyme stability, its expression level in the resistant coliform bacteria or result in a higher turn-over rate of the mutated β-lactamase by facilitating conformational changes.