Emergence of a novel β-lactamase in food of animal origin: characterization of SHV-111, from a cheese isolate of Klebsiella pneumoniae, with an amino acid substitution in a strictly conserved position (P174S) in the Ω-loop

The -loop is a conserved structural element in all class A b-lactamases. Amino acid substitutions in this loop appear to change the conformational flexibility of the catalytic region, resulting in the evolution of b-lactamases. The stepwise accumulation of mutations observed during the emergence of extended-spectrum b-lactamases (ESBLs) makes it highly probable that higher-order combinations of mutations occurred in clinical settings potentially after exposure to antibiotics [1]. One mutation may change the active site topology, another may enhance catalytic activity and some may be compensatory. The current hypotheses for the evolution of b-lactamases are drawn mainly from human clinical studies and in vitro evolution experiments. However, as stated by Martínez et al. [2] ‘an accurate prediction of evolutionary trajectories, all the steps in evolution that are required to produce a given phenotype, requires better measurements and a more complete understanding of all selection pressures in both human and animals, environmental variations and evolutionary constraints that constitute the real determinants for the correct natural evolutionary pathways’. Therefore, understanding the effect of novel amino acid substitutions on the phenotypic characteristics of SHV b-lactamases that keep the catalytic site accessible for even traditional b-lactam antibiotics is of utmost importance. In light of the scarcity of data on the role of non-clinical isolates in the evolution of b-lactamases, we characterized a novel SHV b-lactamase, SHV-111, from a cheese isolate (KP-DC6-2), which was identified during monitoring of traditional Egyptian Domiati cheese for ESBLs using conserved primers [3]. The blaSHV-111 gene was amplified by PCR using the primer pair SHV-F2 (CGGCC TTCACTCAAGGATGTA) and SHV-R2 (GTGCTGCGGGCCGGATAAC). The purified whole PCR fragment of the blaSHV-111 gene containing only the coding region was cloned into the SmaI-digested plasmid vector pBAD33 using the TaKaRa Ligation kit (Takara Bio). This cloning vector has a chloramphenicol resistance gene and the PBAD promoter of the araBAD (arabinose) operon for gene expression. The new recombinant plasmid was named pSHV-111 and used to transform Escherichia coli TG1 for expression of the cloned blaSHV-111. E. coli TG1 expressing the blaSHV-111 gene (TG1/pSHV-111) was phenotypically characterized by the broth microdilution method. The results were interpreted according to CLSI [4] guidelines for an ESBL producer: an MIC value of at least one extendedspectrum cephalosporin (ceftazidime and ceftriaxone) or aztreonam of 2 mg l . Analytical IEF was conducted to detect the isoelectric point (pI) and Southern blot analysis was carried out for localization of this gene. Sequence analysis of the blaSHV-111 gene (GenBank accession number, AB372881) revealed five silent nucleotide substitutions (in comparison with blaSHV-1, GenBank accession number, AF148850) of CfiT, AfiG, GfiA, CfiG and CfiG at positions 357, 402, 705, 759 and 786, respectively. Interestingly, a codon change of CCC to TCC resulted in one amino acid substitution, serine instead of proline at the strictly conserved Ambler position 174, namely, P174S. This amino acid substitution was not enough to widen its substrate profile (Table 1) to meet the criteria of CLSI [4] for ESBL producers. Analysis of the b-lactamase content by IEF showed the presence of a single b-lactamase co-focusing with SHV1 (pI 7.6). Southern hybridization analysis revealed chromosomal localization of this gene (data not shown).