The rise of bacterial resistance to antibiotics is a growing global health crisis. World Health Organization estimated that by 2050 antibiotic resistance could lead to as many as 10 million deaths annually due to infections that are no longer treatable [1]. Multidrug resistant pathogens, such as Stenotrophomonas maltophilia, present a particular challenge due to their resistance to multiple antibiotics. Gram-negative opportunistic pathogen S. maltophilia has been rapidly acquiring resistance, making infections progressively more difficult to treat, while the mechanisms underlying its antibiotic resistance remain understudied. S. maltophilia is known to exhibit resistance to various antibiotics, including β-lactams, which are among the most clinically significant antibiotics [2]. Although S. maltophilia resistance to β-lactam antibiotics is well-documented, only two β-lactamases, L1 (a metallo-β-lactamase) and L2 (a class A serine β-lactamase), have been researched and characterized as enzymes that confer resistance by degrading β-lactam antibiotics [3]. We previously identified that β-lactam resistant S. maltophilia SM3 isolate codes 10 additional proteins that are homologous to known β-lactamases, suggesting that these proteins may also contribute to the β-lactam resistance.
The aim of this study was to evaluate whether the 10 β-lactamase homologous proteins identified in S. maltophilia contribute to β-lactam resistance. To achieve this, a bioinformatic analysis using the Beta-Lactamase DataBase was performed to identify and compare the sequences of analysed proteins to verified β-lactamases from various bacterial species [4]. Then the genes encoding these homologous proteins were cloned into expression plasmids for functional analysis. The enzymatic activity was assessed in an Escherichia coli expression strain using the nitrocefin assay, a standard method for detecting β-lactamase activity. Additionally, the role of these proteins in antibiotic resistance will be assessed by performing a minimum inhibitory concentration analysis.
The bioinformatic analysis revealed that all the proteins examined shared homology with known β-lactamases, with sequence identities ranging from 24.8% to 29.5%. This level of homology is considered sufficient to support further investigation into their potential as functional β-lactamases [5]. The nitrocefin assay, validated with the well-characterized L1 β-lactamase, was successfully applied to three of the homologous proteins. The remaining putative β-lactamases will be evaluated using the same approach with the nitrocefin assay, and additional evaluation methods will be employed to further explore their contribution to β-lactam resistance.