Materials and Methods

Enterococcus faecalisCRISPR-Cas Is a Robust Barrier to Conjugative Antibiotic Resistance Dissemination in the Murine Intestine

Bacteria and reagents used.

Strains used in this study are shown in Table 1. E. faecalis strains were cultured in brain heart infusion (BHI) broth or on BHI agar at 37°C. Antibiotic concentrations used were as follows: rifampin, 50 μg/ml; fusidic acid, 25 μg/ml; spectinomycin, 500 μg/ml; streptomycin, 500 μg/ml; erythromycin, 50 μg/ml. Antibiotics were purchased from Sigma-Aldrich or Research Products International (RPI).

Conjugation experiments.

Donor and recipient strains were cultured overnight in BHI broth in the absence of antibiotic selection. The following day, cultures were diluted 1:10 into fresh BHI and incubated at 37°C for 1.5 h. For planktonic conjugations at a 1:9 donor/recipient ratio, 2 ml of donor and 18 ml of recipient were mixed in a flask and incubated without agitation at 37°C for 30 min to 18 h. For planktonic conjugations at a 1:1 donor/recipient ratio, 10 ml of donor and 10 ml of recipient were mixed in a flask and incubated without agitation at 37°C for 30 min to 18 h. At each time point, 1 ml of the mating reaction mixture was removed and used for serial dilutions and plating on selective media. For biofilm mating reactions at a 1:9 donor/recipient ratio, 100 μl of donor was mixed with 900 μl of recipient, and for mating reactions at a 1:1 donor/recipient ratio, 500 μl of donor was mixed with 500 μl of recipient. The mixture was centrifuged for 1 min at 16,000 × g. After centrifugation, 100 μl supernatant was used to resuspend the pellet, which was then spread-plated on nonselective BHI agar. To allow for sampling of multiple time points of biofilms, multiple identical conjugation reactions were generated using the same donor and recipient inocula. The conjugation reaction mixtures were incubated at 37°C for 30 min to 18 h. At each time point, cells were collected by washing and scraping an agar plate using 2 ml of 1× phosphate-buffered saline (PBS) supplemented with 2 mM EDTA, and serial dilutions were plated on selective media. For all matings, BHI agar supplemented with antibiotics was used to quantify the donor (spectinomycin, streptomycin, and erythromycin), recipient (rifampin and fusidic acid), and transconjugant (rifampin, fusidic acid, and erythromycin) populations. Plates were incubated for 36 to 48 h at 37°C. Plates with 30 to 300 colonies were used to calculate the number of CFU per milliliter.

Mouse model of E. faecalis colonization.

Seven days prior to bacterial colonization, 6- to 8-week-old C57BL/6 mice were gavaged with 100 μl of an antibiotic cocktail (streptomycin 1 mg/ml, gentamicin 1 mg/ml, erythromycin 200 μg/ml), and given a water bottle ad libitum with the same antibiotic cocktail for 6 days following gavage. Twenty-four hours before bacterial inoculation, antibiotic water was removed and replaced with standard sterile antibiotic-free water. Bacteria were grown overnight in BHI, and mice were gavaged with 109 CFU in PBS of each bacterial strain as experimental groups indicated (1:1 donor/recipient ratio). Samples used for gavage were plated on BHI to confirm that inocula were equal across strains. Fecal samples from mice were collected at 0 h, 24 h, 48 h, and 96 h. Fecal samples were resuspended in 1 ml of sterile PBS, and dilutions were plated on BHI agar supplemented with antibiotics to quantify the donor (spectinomycin, streptomycin, and erythromycin), recipient (rifampin and fusidic acid), and transconjugant (rifampin, fusidic acid, and erythromycin) populations. Plates were incubated for 36 to 48 h at 37°C. Plates with 30 to 300 colonies were used to calculate CFU/gram of feces. Experiments were performed in duplicate or triplicate as follows. For E. faecalis OG1SSp pAM714/T11RF (with or without cas9) cocolonization, three independent experiments were performed consisting of 4, 4, and 6 mice per group per experiment. For OG1SSp pAM771/T11RF (with or without cas9) cocolonization, two independent experiments were performed consisting of five mice per group, except in the second experiment where five mice were used for each group (control and wild-type T11RF groups) and six mice were used for the T11RFΔcas9 group. Data from individual experimental replicates were combined and graphed together. All animal protocols were approved by the Institutional Animal Care and Use Committee of the University of Colorado Anschutz Medical Campus (protocol 00253).

Colony PCR to verify in vivo transconjugants.

Fecal pellets were collected at 0 h, 24 h, 48 h, and 96 h, weighed, and resuspended in 1 ml PBS. Portions (20 μl) were plated at multiple dilutions on BHI containing rifampin, fusidic acid, and erythromycin. Individual colonies were picked and resuspended in 20 μl nuclease-free water, and 1 μl was used in PCR with Taq DNA polymerase (New England Biolabs). Primers amplified the repB region of plasmids pAM714 and pAM771 (pAD1 repB-For For stands for forward, 5′-CGT TCC ATG TGT CTA ACA ATT GTA TTA AAC-3′, and pAD1 repB-Rev Rev stands for reverse, 5′-CGA TGA TGG TAG CAA TTC AAG AAG G-3′).

Article TitleEnterococcus faecalisCRISPR-Cas Is a Robust Barrier to Conjugative Antibiotic Resistance Dissemination in the Murine Intestine

Abstract

CRISPR-Cas systems are barriers to horizontal gene transfer (HGT) in bacteria. Little is known about CRISPR-Cas interactions with conjugative plasmids, and studies investigating CRISPR-Cas/plasmid interactions in in vivo models relevant to infectious disease are lacking. These are significant gaps in knowledge because conjugative plasmids disseminate antibiotic resistance genes among pathogens in vivo, and it is essential to identify strategies to reduce the spread of these elements. We use enterococci as models to understand the interactions of CRISPR-Cas with conjugative plasmids. Enterococcus faecalis is a native colonizer of the mammalian intestine and harbors pheromone-responsive plasmids (PRPs). PRPs mediate inter- and intraspecies transfer of antibiotic resistance genes. We assessed E. faecalis CRISPR-Cas anti-PRP activity in the mouse intestine and under different in vitro conditions. We observed striking differences in CRISPR-Cas efficiency in vitro versus in vivo. With few exceptions, CRISPR-Cas blocked intestinal PRP dissemination, while in vitro, the PRP frequently escaped CRISPR-Cas defense. Our results further the understanding of CRISPR-Cas biology by demonstrating that standard in vitro experiments do not adequately model the in vivo antiplasmid activity of CRISPR-Cas. Additionally, our work identifies several variables that impact the apparent in vitro antiplasmid activity of CRISPR-Cas, including planktonic versus biofilm settings, different donor-to-recipient ratios, production of a plasmid-encoded bacteriocin, and the time point at which matings are sampled. Our results are clinically significant because they demonstrate that barriers to HGT encoded by normal (healthy) human microbiota can have significant impacts on in vivo antibiotic resistance dissemination.

IMPORTANCE CRISPR-Cas is a type of immune system in bacteria that is hypothesized to be a natural impediment to the spread of antibiotic resistance genes. In this study, we directly assessed the impact of CRISPR-Cas on antibiotic resistance dissemination in the mammalian intestine and under different in vitro conditions. We observed a robust effect of CRISPR-Cas on in vivo but not in vitro dissemination of antibiotic resistance plasmids in the native mammalian intestinal colonizer Enterococcus faecalis. We conclude that standard in vitro experiments currently do not appropriately model the in vivo conditions where antibiotic resistance dissemination occurs between E. faecalis strains in the intestine. Moreover, our results demonstrate that CRISPR-Cas present in native members of the mammalian intestinal microbiota can block the spread of antibiotic resistance plasmids.


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