KSA-1, a naturally occurring Ambler class A extended spectrum β-lactamase from the enterobacterial species Kosakonia sacchari

Abstract

Background: Several bacterial species belonging to the Gammaproteobacteria possess intrinsic class A β-lactamase genes that may represent a source of further dissemination and acquisition to other Gram-negative species. Here we characterised KSA-1 class A β-lactamase, the gene of which was identified within the chromosome of an environmental Enterobacterales species, namely Kosakonia sacchari, which was also recently identified as the progenitor of an MCR-like colistin-resistance determinant. Methods: In silico analysis using the GenBank database identified a class A β-lactamase gene within the chromosome of K. sacchari SP1 (GenBank accession no. WP_017456759). The corresponding protein KSA-1 shared 63% amino acid identity with the intrinsic CKO-1 from Citrobacter koseri and 53% with TEM-1. Using the K. sacchari DSM 100203 reference strain as a template, blaKSA-1 was amplified, cloned into the plasmid pUCp24 and expressed in Escherchia coli TOP10. Minimal inhibitory concentrations and kinetic parameters were obtained from the purified enzyme. Results: K. sacchari strain SP1 conferred resistance to amino-, carboxy- and ureido-penicillins only. Once produced within E. coli, KSA-1 showed a typical clavulanic acid-inhibited extended spectrum β-lactamase associated with a peculiar temocillin resistance profile. Kinetic assays were performed using a purified extract of KSA-1 and demonstrated a high hydrolysis rate for benzylpenicillin and piperacillin, as well as weakly extended spectrum cephalosporins. Determination of inhibitory constants showed 50% inhibitory concentration values of 2.2, 3 and 1.8 nM for clavulanic acid, tazobactam and avibactam, respectively. Analysis of sequences surrounding the blaKSA-1 gene did not reveal any mobile element that could have been involved in the acquisition of this β-lactamase gene in that species. Conclusion: KSA-1 is a class A extended spectrum β-lactamase distantly related to known extended spectrum or broad-spectrum Ambler class A β-lactamases, which is highly resistant to temocillin. The blaKSA-1 gene could be considered as intrinsic within the species.

Keywords

• ESBL
• KSA-1
• Kosakonia sacchari