Downloaded from www.microbiologyresearch.org by IP: 54.70.40.11 On: Tue, 11 Dec 2018 13:34:55 Emergence of a novel b-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 -loop Ahmed M. Hammad, 1 Toshi Shimamoto 2 and Tadashi Shimamoto 2, * Sir, 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 step- wise accumulation of mutations observed during the emer- gence of extended-spectrum b-lactamases (ESBLs) makes it highly probable that higher-order combinations of muta- tions 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 evo- lution of b-lactamases are drawn mainly from human clini- cal studies and in vitro evolution experiments. However, as stated by Martínez et al. [2] ‘an accurate prediction of evolu- tionary 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, environ- mental variations and evolutionary constraints that consti- tute the real determinants for the correct natural evolutionary pathways’. Therefore, understanding the effect of novel amino acid substitutions on the phenotypic charac- teristics 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 bla SHV-111 gene was amplified by PCR using the primer pair SHV-F2 (CGGCC TTCACTCAAGGATGTA) and SHV-R2 (GTGCTGC- GGGCCGGATAAC). The purified whole PCR fragment of the bla SHV-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 P BAD 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 bla SHV-111 . E. coli TG1 expressing the bla SHV-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 extended- spectrum cephalosporin (ceftazidime and ceftriaxone) or aztreonam of 2 mg l 1 . Analytical IEF was conducted to detect the isoelectric point (pI) and Southern blot analysis was carried out for localization of this gene. Sequence analy- sis of the bla SHV-111 gene (GenBank accession number, AB372881) revealed five silent nucleotide substitutions (in comparison with bla SHV-1 , GenBank accession number, AF148850) of CfiT, AfiG, GfiA, CfiG and CfiG at posi- tions 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 con- served Ambler position 174, namely, P174S. This amino acid substitution was not enough to widen its substrate pro- file (Table 1) to meet the criteria of CLSI [4] for ESBL pro- ducers. Analysis of the b-lactamase content by IEF showed the presence of a single b-lactamase co-focusing with SHV- 1 (pI 7.6). Southern hybridization analysis revealed chromo- somal localization of this gene (data not shown). Interestingly, substitution of the proline at position 174 has never been detected in previously identified SHV b-lacta- mases (http://www.lahey.org/studies/). A previous study [5] found that residue P174 displays the largest structural devia- tion (>0.45 nm) in the -loop that allows the entrance of b- lactam antibiotics to the active site of TEM b-lactamases. Another interesting study [6] conducted mutant selection with chromosomally encoded PenA from Burkholderia Received 22 October 2016; Accepted 30 December 2016 Author affiliations: 1 Department of Food Hygiene and Control, Faculty of Veterinary Medicine, University of Sadat City, Sadat City, Egypt; 2 Laboratory of Food Microbiology and Hygiene, Graduate School of Biosphere Science, Hiroshima University, 1-4-4 Kagamiyama, Higashi-Hiroshima, Hiroshima 739-8528, Japan. *Correspondence: Tadashi Shimamoto, tadashis@hiroshima-u.ac.jp Keywords: b-lactamase; Klebsiella pneumoniae; SHV; cheese. Abbreviation: ESBL, extended-spectrum b-lactamase. CORRESPONDENCE Hammad et al., Journal of Medical Microbiology 2017;66:96–97 DOI 10.1099/jmm.0.000396 000396 ã 2017 The Authors 96