CRISPR-Cas9-mediated IncF plasmid curing in extraintestinal pathogenic Escherichia coli

ABSTRACT IncF plasmids are commonly found in extra-intestinal pathogenic Escherichia coli (ExPEC) strains, serving as reservoirs for antimicrobial resistance (AMR) genes and virulence factors, persistently coexisting with ExPEC lineages. Multidrug-resistant (MDR) high-risk ExPEC clones, particularly ST131, ST1193, and ST410, have acquired diverse IncF plasmids over time, containing various AMR determinants, contributing significantly to their global success. However, the broader roles of these IncF plasmids in the success of MDR ExPEC clones, beyond AMR, remain elusive. In this study, we employed a novel clustered regularly interspaced short palindromic repeats–CRISPR-associated protein-9 nuclease (CRISPR-Cas9)-mediated pCasCure plasmid-curing system to precisely remove specific IncF plasmids among ExPEC clones (ST1193, ST131, and ST410). Antibiotic-resistant parent strains reverted to antibiotic-susceptible states post-curing; however, IncF plasmid curing did not show significant impact on bacterial in vitro growth and had little impact on other in vitro phenotypes, including survival in water, dry environment and biofilm production. In addition, IncF plasmid curing did not affect the conjugation frequency of KPC-producing pKpQIL plasmid. This study represents a pivotal initial step in understanding the precise roles of IncF plasmids in the success of ExPEC. Future research will be crucial in investigating their influence on cell invasion and in vivo fitness, thereby providing a more comprehensive perspective on the functions of IncF plasmids in MDR ExPEC clones. IMPORTANCE Understanding the role of IncF plasmids in the success of drug-resistant bacteria has far-reaching implications for tackling antibiotic resistance. The study's use of a novel CRISPR-Cas9-mediated plasmid-curing system provides a precision tool for dissecting the specific impact of IncF plasmids on ExPEC clones, especially high-risk, multidrug-resistant strains like ST131, ST1193, and ST410. The study offers a crucial stepping stone for future research into understanding how these plasmids influence more complex aspects of bacterial behavior, such as cell invasion and in vivo fitness.

I ncFs are the most abundant plasmid types among extra-intestinal pathogenic Escherichia coli (ExPEC) (1).IncF plasmids are large (>50 kb), mosaic, and con tain antimicrobial resistance (AMR) genes, virulence associated factor genes, various addiction, restriction, and truncated transfer systems (2).The presence of addi tion/restriction systems, combined with truncated transfer regions, has led to IncF plasmid persistence with subsequent fixation within certain ExPEC lineages (3).
The clustered regularly interspaced short palindromic repeats-CRISPR-associated protein-9 nuclease (CRISPR-Cas9) system is a novel tool to generate site-specific double-strand breaks for genome editing in mammalian cells, plants, fungi, and bacteria (8,9).We previously developed a novel CRISPR-Cas9-mediated pCasCure plasmid-curing system that can precisely cure specific antimicrobial genes and plasmids in bacterial hosts (10).Currently, limited information is available on which specific biological features on IncF plasmids have contributed to the success of MDR ExPEC clones (3,7,11).In this study, we employed the pCasCure system to remove specific IncF plasmids among MDR high-risk ExPEC clones belonging to ST1193, ST131, and ST410, to assess the impact of IncF plasmids on ExPEC antimicrobial resistance and in vitro phenotypes.Our study represents a first step in utilizing CRISPR-Cas9-mediated plasmid curing system toward deciphering the roles of IncF plasmids that contribute to the success of ExPEC lineages.
c "-" indicates negative for the genes or plasmids.
curves were generated using various media: Luria-Bertani, M9 minimal medium, artificial urine medium, and Davis-Mingoli medium, employing previously described methods (18).Briefly, overnight cultures were diluted 1:1,000 in the respective media.Subse quently, 150-µL aliquots were dispensed into flat-bottomed 96-well plates.Plates were sealed with breathable membranes and incubated at 37°C.Optical density (OD) at 600 nm was recorded at 10-minute intervals over 24 hours.Survival in water and dry conditions was evaluated with a method described previously, with minor modifications (18).Briefly, overnight cultures were diluted to approximately 10 8 CFU and then inocula ted into 10 mL of autoclaved tap water.Bacterial survival was assessed after 24 and 72 hours of incubation at 37°C through colony plating.For assessing survival in a dry environment, a 96-well flat-bottomed microtiter plate was utilized.Dry bacterial samples were exposed for 2, 5, and 7 hours at room temperature, followed by colony plating.Biofilm formation was evaluated using a 96-well polyvinyl chloride microtiter plate with a 24-hour incubation at 30°C using the crystal violet method (18).All the aforementioned in vitro assays were performed in at least three biological replicates, and each experiment was repeated twice.Growth curve analysis revealed that plasmid curing had no discernible impact on the growth of cured strains in all eight pairs tested, which included Luria-Bertani, M9 minimal medium, artificial urine medium, and Davis-Mingoli medium (data not shown).This suggests that the IncF plasmids had a very limited effect on the growth fitness of the bacterial hosts among these epidemic MDR ExPEC strains.In terms of survival in water, all eight wild-type and IncF plasmid-cured pairs exhibited robust survival rates at 24, 48, and 72 hours, with growth increasing to over 250%-350% at 72 hours.However, no significant difference was observed between the wild-type and plasmid-cured pairs in terms of water survival (data not shown).Similarly, survival in a dry environment showed no significant differences between individual wild-type and plasmid-cured pairs.However, there were some strain-to-strain variations (Fig. S1).For example, ST1193 strain EC16019 displayed approximately 25% survival at 2 hours on a dry plate, while ST131-A strain EC16639, ST131-B strain EC12401, and ST410 strain AZ1114 showed less than 2% growth at 2 hours.Nevertheless, all strains, including both the wild-type and plasmid-cured mutants, exhibited low survival rates (< 3%) after 7 hours of exposure to a dry environment, suggesting that these MDR ExPEC strains do not survive well in environments lacking moisture.Regarding biofilm production, all eight pairs displayed low-level biofilm production [average optical density (OD) = 0.16] compared to the positive control strain, Staphylococcus.epidermidis strain RP62A (OD = 1.96) (Fig. S2).It is noted that IncF plasmid curing in ST131 C2 strain EC16611 and ST410 strain AZ1114 resulted in a significant reduction in biofilm formation compared with their wild-type strain.This suggests that plasmid-borne genes may play a role in the variation of biofilm production among clinical MDR ExPEC isolates.
Lastly, we investigated whether the presence of IncF plasmids had an impact on the acquisition of carbapenemase-producing plasmids.To address this, we conducted plasmid filter mating experiments using the epidemic IncFIIK2 bla KPC -harboring pKpQIL plasmid (pKpQIL-03) (19) as the vector.Diaminopimelic acid auxotrophic E. coli DH10B harboring pKpQIL-03 was used as the donor, transferring the plasmid into various E. coli pairs, except for AZ1114, which already contained a carbapenemase gene bla VIM-1 on an IncN plasmid.Transconjugants were selected on meropenem (2 µg/mL) plates and confirmed through PCR.The conjugation frequency was calculated as the ratio of the number of transconjugants to the number of recipients.The results revealed that the pKpQIL-03 plasmid successfully transferred into these ExPEC pairs, with a high conjugation frequency ranging from 10 −3 to 10 −2 (data not shown).However, there were no significant conjugation frequency differences observed between the wild-type and IncF plasmid-cured mutants.The results suggest that, despite both belonging to the same category of IncF plasmids, the IncF plasmids from clinical MDR ExPEC strains did not impede the acquisition of IncFIIK pKpQIL plasmids.This implies that IncF plasmids in MDR ExPEC strains might be compatible with IncFIIK pKpQIL plasmids.
In summary, this study represents one of the first investigations utilizing a precise CRISPR-Cas plasmid curing platform to specifically probe the role of IncF plasmids in MDR ExPEC strains.Our results clearly demonstrate that the successful removal of resistance plasmids can sensitize the bacteria to antibiotics once more.Considering the strong association between IncF plasmids and MDR ExPEC strains, we found that it is imperative to delve deeper into the functions of IncF plasmids beyond their involvement in antimicrobial resistance.One noteworthy discovery is that the IncF plasmid has a limited impact on the in vitro fitness of ExPEC hosts.This suggests that these plasmids may have well adapted with these bacterial hosts.To gain a more comprehensive understanding of the IncF plasmid's role, further research is necessary to investigate its influence on cell invasion and in vivo fitness.This approach will provide an accurate characterization of how IncF plasmids contribute to the success of epidemic MDR ExPEC clones.Such data will aid in developing novel treatment strategies and provide novel genomic surveillance targets to track emerging MDR ExPEC clones.Future studies will use the same curing strategies to investigate the role of IncF plasmids among other ExPEC MDR clones (ST38, ST167, ST405, and ST648) and non-MDR high-risk ExPEC clones (ST69, ST73, and ST95).

FIG 1
FIG 1 Comparative analysis of IncF plasmids cured in ExPEC strains.Light blue shading represents shared regions of homology, while light green shading indicates reversed displayed homology.Open reading frames (ORFs) are depicted as arrows and colored according to predicted gene function.Orange arrows represent plasmid scaffold regions.Genes associated with the tra locus are indicated by green arrows, and replication-associated genes are denoted by dark blue arrows.Antimicrobial resistance genes are highlighted with red arrows, while accessory genes are marked with yellow arrows.