Engineering conjugative CRISPR-Cas9 systems for the targeted control of enteric pathogens and antibiotic resistance

Abstract

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.

Fig 1. Maps of the self-transmissible conjugative plasmids carrying CRISPR-Cas9 antimicrobials.

(A) Plasmid pRK24-CRISPR was engineered to carry Cas9 and gRNAeae targeting eae-positive pathogenic E. coli. (B) Plasmid pTF16, a derivative of pBP136, contains Cas9 and dual gRNAs (gRNAeae and gRNAssaN) targeting pathogenic E. coli (EHEC and EPEC) and S. enterica. (C) Plasmid pTF18, a derivative of pBP136, contains Cas9 and dual gRNAs (gRNAssaN and gRNA gRNAbla_CMY-2) that targeting S. enterica and bla_CMY-2 plasmids.

Fig 2. Conjugation with pRK24-CRISPR kills eae⁺ and not Δeae E. coli O157:H7.

A triparental mating assay was performed to evaluate the effect of pRK24-CRISPR conjugation to the targeted O157:H7 strain 905Δtir and the non-targeted strain 905Δeae. The numbers of the recipient bacteria and the transconjugants were enumerated following 6 h mating done in triplicate. Error bars represent the geometric mean log values of bacterial counts plus the geometric standard deviation. ** P<0.001 by the Student’s t-test.

Fig 3. Conjugation of pTF16 simultaneously kills EPEC and S. enterica.

In the triparental mating assays, EPEC O128:H2 (Nal^R) and S. enterica ser Typhimurium TR5877 Rif^R) mated with EC100 (Cm^R) strains harboring pBP136kan or pTF16. The numbers of the recipient cells were enumerated after 6 h mating. Error bars represent the geometric mean of log values of bacterial counts plus the geometric standard deviation. * P < 0.001.

Fig 4. Conjugation of pTF18 eliminates bla_CMY-2 plasmids from the host E. coli.

E. coli C3b harboring pAR60302 (Amp^R, Cm^R) was mating with EC100 pTF18 or pTF16. Elimination efficiency is the percentage of the recipient cells that lost Amp resistance following the mating. Data points represent the mean value plus the geometric standard deviation. * p<0.05.

Fig 5. CRISPR-Cas9 antimicrobial delivered by pTF16 reduces the burden of EPEC O55:H6 in the murine intestine.

Neonate mice were orally infected with 10⁵ CFU EPEC. On day 1 and 2 post-infection, the pups in three groups were treated with E. coli MF1 pBP136kan, E. coli MF1 pTF16, or PBS, respectively. Small intestine and colon tissues were collected, homogenized and plated for bacterial counts. E+ indicates samples positive for EPEC O55:H6 only by an enrichment procedure. Error bars represent the geometric mean plus the geometric standard deviation. Significance is shown via grouping: a = significantly different from groups marked with b or c; b = significantly different from groups marked with a or c; c = significantly different from groups marked with a or b. * p ≤ 0.05.