Many clinical species of bacteria were isolated from biological samples such as urines, blood and wound in Saint Camille medical centre of Ouagadougou. Among the concerned species, the most important members were Escherichia coli and Klebsiella pneumoniae. These β-lactamases producing isolates were directly screened by PCR to identify the nature of the amplified genes responsible for penicillin destroying activity. Therefore specific TEM and SHV primers were used. The PCR products were sequenced. The sequencing results indicated that the parental forms blaTEM-1 and blaSHV-1 were the most common determinants of β-lactamase found, respectively in Escherichia species and Klebsiella pneumoniae. The bacterial susceptibility analysis by MICs measurement clearly correlated the presence of concerned β-lactamase determinants and their resistance patterns. This study is part of a set of investigations carried out by our laboratory to assess the β-lactamase incidence in the failure of β-lactam therapy. In particular, the purpose of this study was to determine the precise nature of β-lactamase supporting the low susceptibility of host bacteria towards penicillins.