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Conjugative Delivery of CRISPR-Cas9 for the Selective Depletion of Antibiotic-Resistant Enterococci.

ABSTRACT: The innovation of new therapies to combat multidrug-resistant (MDR) bacteria is being outpaced by the continued rise of MDR bacterial infections. Of particular concern are hospital-acquired infections (HAIs) that are recalcitrant to antibiotic therapies. The Gram-positive intestinal pathobiont Enterococcus faecalis is associated with HAIs, and some strains are MDR. Therefore, novel strategies to control E. faecalis populations are needed. We previously characterized an E. faecalis type II CRISPR-Cas system and demonstrated its utility in the sequence-specific removal of antibiotic resistance determinants. Here, we present work describing the adaption of this CRISPR-Cas system into a constitutively expressed module encoded on a pheromone-responsive conjugative plasmid that efficiently transfers to E. faecalis for the selective removal of antibiotic resistance genes. Using in vitro competition assays, we show that these CRISPR-Cas-encoding delivery plasmids, or CRISPR-Cas antimicrobials, can reduce the occurrence of antibiotic resistance in enterococcal populations in a sequence-specific manner. Furthermore, we demonstrate that deployment of CRISPR-Cas antimicrobials in the murine intestine reduces the occurrence of antibiotic-resistant E. faecalis by several orders of magnitude. Finally, we show that E. faecalis donor strains harboring CRISPR-Cas antimicrobials are immune to uptake of antibiotic resistance determinants in vivo Our results demonstrate that conjugative delivery of CRISPR-Cas antimicrobials may be adaptable for future deployment from probiotic bacteria for exact targeting of defined MDR bacteria or for precision engineering of polymicrobial communities in the mammalian intestine.

SUBMITTER: Rodrigues M

PROVIDER: S-EPMC6811441 | biostudies-literature | 2019 Nov

REPOSITORIES: biostudies-literature

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Conjugative Delivery of CRISPR-Cas9 for the Selective Depletion of Antibiotic-Resistant Enterococci.

Rodrigues Marinelle M McBride Sara W SW Hullahalli Karthik K Palmer Kelli L KL Duerkop Breck A BA

Antimicrobial agents and chemotherapy 20191022 11

The innovation of new therapies to combat multidrug-resistant (MDR) bacteria is being outpaced by the continued rise of MDR bacterial infections. Of particular concern are hospital-acquired infections (HAIs) that are recalcitrant to antibiotic therapies. The Gram-positive intestinal pathobiont Enterococcus faecalis is associated with HAIs, and some strains are MDR. Therefore, novel strategies to control E. faecalis populations are needed. We previously characterized an E. faecal ...[more]

PMID: 31527030

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Project description:Pathogenic Escherichia coli and Salmonella enterica pose serious public health threats due to their ability to cause severe gastroenteritis and life-threatening sequela, particularly in young children. Moreover, the emergence and dissemination of antibiotic resistance in these bacteria have complicated control of infections. Alternative strategies that effectively target these enteric pathogens and negate or reduce the need of antibiotics are urgently needed. Such an alternative is the CRISPR-Cas9 system because it can generate sequence-specific lethal double stranded DNA breaks. In this study, two self-transmissible broad host range conjugative plasmids, pRK24 and pBP136, were engineered to deliver multiplexed CRSIPR-Cas9 systems that specifically target Enterohemorrhagic and Enteropathogenic strains of E. coli (EHEC and EPEC), S. enterica, and blaCMY-2 antibiotic resistance plasmids. Using in vitro mating assays, we show that the conjugative delivery of pRK24-CRISPR-Cas9 carrying guide RNAs to the EPEC/EHEC eae (intimin) gene can selectively kill enterohemorrhagic E. coli O157 eae+ cells (3 log kill at 6 h) but does not kill the isogenic Δeae mutant (P<0.001). Similar results were also obtained with a pBP136 derivative, pTF16, carrying multiplexed guide RNAs targeting E. coli eae and the S. enterica ssaN gene coding for the type III secretion ATPase. Another pBP136 derivative, TF18, carries guide RNAs targeting S. enterica ssaN and the antibiotic resistance gene, blaCMY-2, carried on the multi-drug resistant pAR06302. Introduction of pTF18 into bacteria harboring pAR06302 showed plasmids were cured at an efficiency of 53% (P<0.05). Using a murine neonate EPEC infection model, pTF16 was delivered by a murine derived E. coli strain to EPEC infected mice and showed significant reductions of intestinal EPEC (P<0.05). These results suggest that establishing conjugative CRISPR-Cas9 antimicrobials in the intestinal microbiome may provide protection from enteric pathogens and reduce antibiotic resistance without disrupting the normal microbiota.

Dataset Information

Conjugative Delivery of CRISPR-Cas9 for the Selective Depletion of Antibiotic-Resistant Enterococci.

Publications

Conjugative Delivery of CRISPR-Cas9 for the Selective Depletion of Antibiotic-Resistant Enterococci.

Similar Datasets

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