Engineering probiotic Escherichia coli Nissle 1917 to block transfer of multiple antibiotic resistance genes by exploiting a type I CRISPR-Cas system

ABSTRACT Many multidrug-resistant (MDR) bacteria have evolved through the accumulation of antibiotic resistance genes (ARGs). Although the potential risk of probiotics as reservoirs of ARGs has been recognized, strategies for blocking the transfer of ARGs while using probiotics have rarely been explored. The probiotic Escherichia coli Nissle 1917 (EcN) has long been used for treating intestinal diseases. Here, we demonstrate frequent transfer of ARGs into EcN both in vitro and in vivo , raising concerns about its potential risk of accumulating antibiotic resistance. Given that no CRISPR-Cas system was found in natural EcN, we integrated the type I-E CRISPR-Cas3 system derived from E. coli BW25113 into EcN. The engineered EcN was able to efficiently cleave multiple ARGs [i.e., mcr-1 , bla NDM-1 , and tet (X)] encoding enzymes for degrading last-resort antibiotics. Through co-incubation of EcN expressing Cas3-Cascade and that expressing Cas9, we showed that the growth of the former strain outcompeted the latter strain, demonstrating a better clinical application prospect of EcN expressing the type I-E CRISPR-Cas3 system. In the intestine of a model animal (i.e., zebrafish), the engineered EcN exhibited immunity against the transfer of CRISPR-targeted ARGs. Our work equips EcN with immunity against the transfer of multiple ARGs by exploiting the exogenous type I-E CRISPR-Cas3 system, thereby reducing the risk of the spread of ARGs while using it as a probiotic chassis for generating living therapeutics. IMPORTANCE To reduce the development of antibiotic resistance, probiotics have been considered as a substitute for antibiotics. However, probiotics themselves are reservoirs of antibiotic resistance genes (ARGs). This study introduces a new strategy for limiting the spread of ARGs by engineering the typical probiotic strain Escherichia coli Nissle 1917 (EcN), which has been used for treating intestinal diseases and developed as living therapeutics. We also demonstrate that the type I CRISPR-Cas system imposes a lower growth burden than the type II CRISPR-Cas system, highlighting its promising clinical application potential. Our work not only provides a new strategy for restricting the transfer of ARGs while using probiotics but also enriches the genetic engineering toolbox of EcN, paving the way for the safe use and development of probiotics as living therapeutics.