Phage-delivered sensitisation with subsequent antibiotic treatment reveals sustained effect against antimicrobial resistant bacteria

Liu, Hongbo; Hao, Li; Yuan, Long; Du, Xinying; Yang, Chaojie; Yang, Lang; Xie, Jing; Zhao, Rongtao; Tong, Yigang; Qiu, Shaofu; Song, Hongbin

doi:10.7150/thno.42573

Cited by 20 publications

(16 citation statements)

References 45 publications

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“…Interestingly, the SG18 cells co-electroporated with two spacer RNAs (pCas9_g and pCas9_g*) targeting distinct regions in the SpvB gene did not show any amplification through the colony PCR ( Supplementary Figure S4 ), which suggests that the double-stranded break produced at two distinct positions in the SpvB gene may not induce effective recombination to achieve the double-stranded break repair, hence it degraded the plasmid ( Wang et al, 2016 ). However, the plasmid isolated from the same colonies showed SpvB amplification, which suggests that, the uncut plasmid replicated to restore their copies, which is contrary to a previous study that claimed gene deletion from plasmid was confirmed through negative colony PCR ( Liu et al, 2020 ). Our results suggest that screening the bacterial colonies for plasmid editing solely through colony PCR may give false-positive results.…”

Section: Resultscontrasting

confidence: 98%

CRISPR/Cas9-Based Deletion of SpvB Gene From Salmonella gallinarum Leads to Loss of Virulence in Chicken

Basit

Tahir

Haider

et al. 2022

Front. Bioeng. Biotechnol.

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Salmonella Gallinarum causes fowl typhoid in poultry leading to a huge economic loss to the poultry industry. The large virulence plasmid of S. gallinarum has been associated with various systemic infections in poultry. A five-gene spanning region (spvRABCD) of 7.8 kb on the large plasmid mainly confers virulence to the bacteria. However, the exact role of these genes in virulence has not been elucidated yet. SpvB exhibits delayed cell death by preventing actin polymerization followed by apoptosis during intracellular infection. The specific role of SpvB in causing the disease is not known yet. In the current study, the SpvB gene was deleted through CRISPR/Cas9 method from a large virulent plasmid of locally isolated S. gallinarum strain (SG18). The homology-directed repair method was used for complete deletion of SpvB gene using the modified pCas9 plasmid. The SpvB-deleted S. gallinarum strain (ΔSpvB_SG18), when tested for its virulence in broiler chicken showed no diseases signs and mortality. In addition, the avirulent strain does not affect the bird’s weight and was rapidly cleared from the liver after infection. However, it cleared from the intestine only after 4–5 days, which suggests that the ΔSpvB_SG18 strain is unable to invade from the intestine to the liver. This is the first study to report a complete gene deletion from the S. gallinarum virulent plasmid and its effect. This method will be useful for the deletion of virulent genes from S. gallinarum, to study their role in pathogenesis, and to prepare an effective vaccine strain for controlling fowl typhoid in poultry.

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Section: Resultscontrasting

confidence: 98%

CRISPR/Cas9-Based Deletion of SpvB Gene From Salmonella gallinarum Leads to Loss of Virulence in Chicken

Basit

Tahir

Haider

et al. 2022

Front. Bioeng. Biotechnol.

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“…Furthermore, phages and nanoparticles can be effectively used for the delivery of the CRISPR-Cas9 system into the bacterial pathogens. 13 , 40 In addition to plasmid elimination, the CRISPR-Cas9 could further provide immunity in E. coli against the acquisition of plasmid-mediated ARGs. 14 , 34 …”

Section: Discussionmentioning

confidence: 99%

Targeted Elimination of blaNDM-5 Gene in Escherichia coli by Conjugative CRISPR-Cas9 System

Li¹,

Wan²,

Zhao³

et al. 2022

IDR

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Purpose Plasmid-borne carbapenem resistance gene bla NDM-5 accelerates the dissemination of carbapenem-resistant Enterobacteriaceae. To efficiently eliminate the bla NDM-5 -harboring plasmid and sensitize the antibiotic-resistant bacteria to meropenem, we used the CRISPR-Cas9 system for combating the carbapenem-resistant Escherichia coli ( E. coil ). Methods A series of CRISPR-Cas9 plasmids was constructed, and specific guide RNAs(sgRNA) were designed to target the bla NDM-5 gene. We used chemically transformation or conjugation delivery methods, and the elimination efficiency in each recipient strains was evaluated by plate counting, PCR and quantitative real-time PCR (qPCR). Antimicrobial susceptibility test was carried out by using the broth microdilution method. In addition, we assessed the effect of the CRISPR-Cas9 system of adaptive immunity on the prevention of the exogenous resistant plasmids pNDM-5 by introducing the system into E coli J53. Results The results showed that pCas9, pCas9-oriT and pBAD-Cas9-oriT can effectively eliminate bla NDM-5 in E. coli with >94.00% elimination efficiency. The bla NDM-5 -harboring E. coli successfully restored their susceptibility to meropenem, with eight-fold reduction of minimum inhibitory concentration (MIC) values (from 16 µg/mL to 0.06 µg/mL). The E. coli J53 strain containing plasmid pCas9-N reduced the number of transconjugants by 26-fold. Conclusion The CRISPR-Cas9 system achieved plasmid clearance and simultaneous re-sensitization to meropenem in E. coli . The CRISPR-Cas9 system could block the horizontal transfer of plasmid pNDM-5. The conjugative delivery of CRISPR-Cas9 provides a new tool for the removal of resistance plasmids and sensitize the recipient to carbapenem. It provides a therapeutic approach to counteract the propagation of bla NDM-5 gene among clinical pathogens.

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“…61 Recently, one such system was devised that allows onestep incorporation of the CRISPR-Cas9 system into a lysogenic phage. 62 The strategy encompasses a suicide vector having an inducible suicide gene sacB and a 30-bp target sequence with a PAM motif. This is introduced into a host along with a prophage and another vector facilitating homologous recombination.…”

Section: Crispr-assisted Phage Engineeringmentioning

confidence: 99%

Phage engineering and phage‐assisted CRISPR‐Cas delivery to combat multidrug‐resistant pathogens

Khambhati

Bhattacharjee

Gohil

et al. 2022

Bioengineering & Transla Med

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Antibiotic resistance ranks among the top threats to humanity. Due to the frequent use of antibiotics, society is facing a high prevalence of multidrug resistant pathogens, which have managed to evolve mechanisms that help them evade the last line of therapeutics. An alternative to antibiotics could involve the use of bacteriophages (phages), which are the natural predators of bacterial cells. In earlier times, phages were implemented as therapeutic agents for a century but were mainly replaced with antibiotics, and considering the menace of antimicrobial resistance, it might again become of interest due to the increasing threat of antibiotic resistance among pathogens. The current understanding of phage biology and clustered regularly interspaced short palindromic repeats (CRISPR) assisted phage genome engineering techniques have facilitated to generate phage variants with unique therapeutic values. In this review, we briefly explain strategies to engineer bacteriophages. Next, we highlight the literature supporting CRISPR-Cas9-assisted phage engineering for effective and more specific targeting of bacterial pathogens. Lastly, we discuss techniques that either help to increase the fitness, specificity, or lytic ability of bacteriophages to control an infection.

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Phage-delivered sensitisation with subsequent antibiotic treatment reveals sustained effect against antimicrobial resistant bacteria

Cited by 20 publications

References 45 publications

CRISPR/Cas9-Based Deletion of SpvB Gene From Salmonella gallinarum Leads to Loss of Virulence in Chicken

CRISPR/Cas9-Based Deletion of SpvB Gene From Salmonella gallinarum Leads to Loss of Virulence in Chicken

Targeted Elimination of blaNDM-5 Gene in Escherichia coli by Conjugative CRISPR-Cas9 System

Phage engineering and phage‐assisted CRISPR‐Cas delivery to combat multidrug‐resistant pathogens

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