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

ABSTRACT

BACKGROUND: Several bacterial species belonging to the Gammaproteobacteria possess intrinsic class A ß-lactamase genes that may represent the source of further dissemination and acquisition to other Gram-negative species. Her,e we characterized the KSA-1 class A ß-lactamase which gene was identified into the chromosome of an environmental Enterobacterales species, namely Kosakonia sacchari, recently identified also progenitor of an MCR-like colistin resistance determinant. METHODS: In-silico analysis using the GenBank database identified a class A ß-lactamase gene in the chromosome of Kosakonia sacchari SP1 (GenBank accession no. WP_065368351). The corresponding protein KSA-1 shared 63% amino-acid identity with the intrinsic CKO-1 from Citrobacter koseri and 53% with TEM-1. Using K. sacchari DSM 100203 reference strain as template, the blaKSA-1 was amplified, cloned into plasmid pUCp24 and expressed in E. coli TOP10. MICs and kinetic parameters were obtained from the purified enzyme. RESULTS: Strain K. sacchari SP1 conferred resistance to amino-, carboyx- and ureidopenicillins only. Once produced in E. coli, KSA-1 showed a typical clavulanic-acid inhibited extended-spectrum ß-lactamase (ESBL) associated to a peculiar temocillin resistance profile. Kinetic assays were performed using a purified extract of KSA-1, showed a high hydrolysis rate for benzylpenicillin and piperacillin and weakly extended-spectrum cephalosporins. Determination of inhibitory constants showed IC50 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 ß-lactamase gene in that species. CONCLUSION: KSA-1 is a class A ESBL, distantly related to known ESBLs, with a high activity also temocillin. The blaKSA-1 gene can be considered as intrinsic in the species.