Carbapenems are β-lactam antibiotics that are used as last resort drugs to treat multidrug resistant bacterial infections. Hence, the increasing rate of carbapenem-resistant bacteria within healthcare environments is an issue of concern that needs urgent attention. This resistance is driven by metallo-β-lactamases (MBLs), which can catalyse the hydrolysis of almost all clinically available β-lactams and are resistant to all the available β-lactamase inhibitors.
Genomic analysis of multidrug resistant isolates of the hospital-acquired pathogen, Chryseobacterium indologenes recovered from healthcare-associated wastewater revealed two MBLs, IND-2 and an uncharacterized MBL (CIM-1). As C. indologenes is intrinsically resistant to colistin, very few treatment options remain for isolates that are carbapenem-resistant necessitating an investigation into MBL-driven resistance.
Sequence analysis predicted CIM-1 to be a lipoprotein with an atypical lipobox when compared to all other thus far identified lipoproteins. This is only the second MBL, after the critically important NDM-1, experimentally shown to be membrane associated.
Here we report on the preliminary characterization of CIM-1. Full-length CIM-1, IND-2, and variants thereof, including signal peptide exchanged mutants were cloned and transformed into Escherichia coli C41(DE3), where their resistance profiles were investigated. Soluble CIM-1 conferred high level resistance to β-lactam antibiotics.
Expression of CIM-1 followed by cellular fractionation revealed that this novel MBL was localized to the membrane, supporting predictions that it functions as a lipoprotein. Our hypothesis that cellular localization plays a role in activity was confirmed by the activity profiles of the wild-type CIM-1 and variants. Following protein purification, enzyme kinetics were assessed. Results showed that CIM-1 and IND-2 efficiently hydrolyse all carbapenems tested with CIM-1 having a much higher affinity for substrates compared to IND-2.
This study presents a novel and potent MBL which carries an unusual lipobox not previously reported in other Gram-negative bacteria and highlights the importance of further research into lipidated MBLs.