Conjugative delivery of CRISPR-Cas9 for the selective depletion of antibiotic-resistant enterococci

AbstractThe 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) recalcitrant to antibiotic therapies. The Gram-positive intestinal pathobiontEnterococcus faecalisis associated with HAIs and some strains are MDR. Therefore, novel strategies to controlE. faecalispopulations are needed. We previously characterized anE. faecalisType 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 toE. faecalisfor the selective removal of antibiotic resistance genes. Usingin vitrocompetition 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-resistantE. faecalisby several orders of magnitude. Finally, we show thatE. faecalisdonor strains harboring CRISPR-Cas antimicrobials are immune to uptake of antibiotic resistance determinantsin 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.ImportanceCRISPR-Cas nucleic acid targeting systems hold promise for the amelioration of multidrug-resistant enterococci, yet the utility of such tools in the context of the intestinal environment where enterococci reside is understudied. We describe the development of a CRISPR-Cas antimicrobial, deployed on a conjugative plasmid, for the targeted removal of antibiotic resistance genes from intestinalEnterococcus faecalis. We demonstrate that CRISPR-Cas targeting reduces antibiotic resistance ofE. faecalisby several orders of magnitude in the intestine. Although barriers exist that influence the penetrance of the conjugative CRISPR-Cas antimicrobial among target recipientE. faecaliscells, the removal of antibiotic resistance genes inE. faecalisupon uptake of the CRISPR-Cas antimicrobial is absolute. In addition, cells that obtain the CRISPR-Cas antimicrobial are immunized against the acquisition of new antibiotic resistance genes. This study suggests a potential path toward plasmid based CRISPR-Cas therapies in the intestine.

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

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01454-19 ◽

2019 ◽

Vol 63 (11) ◽

Cited By ~ 5

Author(s):

Marinelle Rodrigues ◽

Sara W. McBride ◽

Karthik Hullahalli ◽

Kelli L. Palmer ◽

Breck A. Duerkop

Keyword(s):

Antibiotic Resistance ◽

Bacterial Infections ◽

Antibiotic Resistance Genes ◽

Multidrug Resistant ◽

Conjugative Plasmid ◽

Antibiotic Resistant ◽

Content Type ◽

Resistance Determinants

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.

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Virulence-determinants and antibiotic-resistance genes of MDR-E. coli isolated from secondary infections following FMD-outbreak in cattle

Scientific Reports ◽

10.1038/s41598-020-75914-9 ◽

2020 ◽

Vol 10 (1) ◽

Author(s):

Abdelazeem M. Algammal ◽

Helal F. Hetta ◽

Gaber E. Batiha ◽

Wael N. Hozzein ◽

Waleed M. El Kazzaz ◽

...

Keyword(s):

Antibiotic Resistance ◽

Resistance Genes ◽

Bacterial Infections ◽

Antibiotic Resistance Genes ◽

Multidrug Resistant ◽

Virulence Determinants ◽

E Coli ◽

Secondary Infections ◽

Random Samples

AbstractThis study aimed to evaluate the prevalence, multidrug-resistance traits, PCR-detection of virulence, and antibiotic-resistance genes of E. coli isolated from secondary infections following FMD-outbreak in cattle. A total of 160 random samples were gathered from private dairy farms in Damietta Province, Egypt. The specimens were subjected to bacteriological examination, serotyping, congo-red binding assay, antibiogram-testing, and PCR-monitoring of virulence-determinant genes (tsh, phoA, hly, eaeA, sta, and lt) as well as the antibiotic-resistance genes (blaTEM, blaKPC, and blaCTX). The prevalence of E. coli was 30% (n = 48) distributed in 8 serogroups (40/48, 83.3%), while 8 isolates (8/48, 16.6%) were untypable. Besides, 83.3% of the examined isolates were positive for CR-binding. The tested strains harbored the virulence genes phoA, hly, tsh, eaeA, sta, and lt with a prevalence of 100% and 50%, 45.8%, 25%, 8.4%, and 6.2%, respectively. Furthermore, 50% of the recovered strains were multidrug-resistant (MDR) to penicillins, cephalosporins, and carbapenems, and are harboring the blaTEM, blaCTX, and blaKPC genes. Moreover, 25% of the examined strains are resistant to penicillins, and cephalosporins, and are harboring the blaTEM and blaCTX genes. To the best of our knowledge, this is the first report concerning the E. coli secondary bacterial infections following the FMD-outbreak. The emergence of MDR strains is considered a public health threat and indicates complicated treatment and bad prognosis of infections caused by such strains. Colistin sulfate and levofloxacin have a promising in vitro activity against MDR-E. coli.

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CRISPR-Cas and Restriction-Modification Act Additively against Conjugative Antibiotic Resistance Plasmid Transfer in Enterococcus faecalis

mSphere ◽

10.1128/msphere.00064-16 ◽

2016 ◽

Vol 1 (3) ◽

Cited By ~ 43

Author(s):

Valerie J. Price ◽

Wenwen Huo ◽

Ardalan Sharifi ◽

Kelli L. Palmer

Keyword(s):

Antibiotic Resistance ◽

High Risk ◽

Enterococcus Faecalis ◽

Resistance Genes ◽

Treatment Options ◽

Antibiotic Resistance Genes ◽

Multidrug Resistant ◽

Content Type ◽

New Genes ◽

Restriction Modification

ABSTRACT Enterococcus faecalis is a bacterium that normally inhabits the gastrointestinal tracts of humans and other animals. Although these bacteria are members of our native gut flora, they can cause life-threatening infections in hospitalized patients. Antibiotic resistance genes appear to be readily shared among high-risk E. faecalis strains, and multidrug resistance in these bacteria limits treatment options for infections. Here, we find that CRISPR-Cas and restriction-modification systems, which function as adaptive and innate immune systems in bacteria, significantly impact the spread of antibiotic resistance genes in E. faecalis populations. The loss of these systems in high-risk E. faecalis suggests that they are immunocompromised, a tradeoff that allows them to readily acquire new genes and adapt to new antibiotics. Enterococcus faecalis is an opportunistic pathogen and a leading cause of nosocomial infections. Conjugative pheromone-responsive plasmids are narrow-host-range mobile genetic elements (MGEs) that are rapid disseminators of antibiotic resistance in the faecalis species. Clustered regularly interspaced short palindromic repeat (CRISPR)-Cas and restriction-modification confer acquired and innate immunity, respectively, against MGE acquisition in bacteria. Most multidrug-resistant E. faecalis isolates lack CRISPR-Cas and possess an orphan locus lacking cas genes, CRISPR2, that is of unknown function. Little is known about restriction-modification defense in E. faecalis. Here, we explore the hypothesis that multidrug-resistant E. faecalis strains are immunocompromised. We assessed MGE acquisition by E. faecalis T11, a strain closely related to the multidrug-resistant hospital isolate V583 but which lacks the ~620 kb of horizontally acquired genome content that characterizes V583. T11 possesses the E. faecalis CRISPR3-cas locus and a predicted restriction-modification system, neither of which occurs in V583. We demonstrate that CRISPR-Cas and restriction-modification together confer a 4-log reduction in acquisition of the pheromone-responsive plasmid pAM714 in biofilm matings. Additionally, we show that the orphan CRISPR2 locus is functional for genome defense against another pheromone-responsive plasmid, pCF10, only in the presence of cas9 derived from the E. faecalis CRISPR1-cas locus, which most multidrug-resistant E. faecalis isolates lack. Overall, our work demonstrated that the loss of only two loci led to a dramatic reduction in genome defense against a clinically relevant MGE, highlighting the critical importance of the E. faecalis accessory genome in modulating horizontal gene transfer. Our results rationalize the development of antimicrobial strategies that capitalize upon the immunocompromised status of multidrug-resistant E. faecalis. IMPORTANCE Enterococcus faecalis is a bacterium that normally inhabits the gastrointestinal tracts of humans and other animals. Although these bacteria are members of our native gut flora, they can cause life-threatening infections in hospitalized patients. Antibiotic resistance genes appear to be readily shared among high-risk E. faecalis strains, and multidrug resistance in these bacteria limits treatment options for infections. Here, we find that CRISPR-Cas and restriction-modification systems, which function as adaptive and innate immune systems in bacteria, significantly impact the spread of antibiotic resistance genes in E. faecalis populations. The loss of these systems in high-risk E. faecalis suggests that they are immunocompromised, a tradeoff that allows them to readily acquire new genes and adapt to new antibiotics.

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Antibiotics in Food Chain: The Consequences for Antibiotic Resistance

Antibiotics ◽

10.3390/antibiotics9100688 ◽

2020 ◽

Vol 9 (10) ◽

pp. 688

Author(s):

Shashi B. Kumar ◽

Shanvanth R. Arnipalli ◽

Ouliana Ziouzenkova

Keyword(s):

Public Health ◽

Antibiotic Resistance ◽

Food Chain ◽

Bacterial Infections ◽

Antibiotic Resistance Genes ◽

Multidrug Resistant ◽

Resistant Bacteria ◽

Alternative Strategies ◽

Antibiotic Resistant ◽

Continuous Use

Antibiotics have been used as essential therapeutics for nearly 100 years and, increasingly, as a preventive agent in the agricultural and animal industry. Continuous use and misuse of antibiotics have provoked the development of antibiotic resistant bacteria that progressively increased mortality from multidrug-resistant bacterial infections, thereby posing a tremendous threat to public health. The goal of our review is to advance the understanding of mechanisms of dissemination and the development of antibiotic resistance genes in the context of nutrition and related clinical, agricultural, veterinary, and environmental settings. We conclude with an overview of alternative strategies, including probiotics, essential oils, vaccines, and antibodies, as primary or adjunct preventive antimicrobial measures or therapies against multidrug-resistant bacterial infections. The solution for antibiotic resistance will require comprehensive and incessant efforts of policymakers in agriculture along with the development of alternative therapeutics by experts in diverse fields of microbiology, biochemistry, clinical research, genetic, and computational engineering.

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Characterization of antibiotic resistance genes in the species of the rumen microbiota

Nature Communications ◽

10.1038/s41467-019-13118-0 ◽

2019 ◽

Vol 10 (1) ◽

Cited By ~ 9

Author(s):

Yasmin Neves Vieira Sabino ◽

Mateus Ferreira Santana ◽

Linda Boniface Oyama ◽

Fernanda Godoy Santos ◽

Ana Júlia Silva Moreira ◽

...

Keyword(s):

Antibiotic Resistance ◽

Resistance Genes ◽

Animal Health ◽

Antibiotic Resistance Genes ◽

Tetracycline Resistance ◽

Multidrug Resistant ◽

Resistant Bacteria ◽

Ruminal Bacteria ◽

Genes Encoding

AbstractInfections caused by multidrug resistant bacteria represent a therapeutic challenge both in clinical settings and in livestock production, but the prevalence of antibiotic resistance genes among the species of bacteria that colonize the gastrointestinal tract of ruminants is not well characterized. Here, we investigate the resistome of 435 ruminal microbial genomes in silico and confirm representative phenotypes in vitro. We find a high abundance of genes encoding tetracycline resistance and evidence that the tet(W) gene is under positive selective pressure. Our findings reveal that tet(W) is located in a novel integrative and conjugative element in several ruminal bacterial genomes. Analyses of rumen microbial metatranscriptomes confirm the expression of the most abundant antibiotic resistance genes. Our data provide insight into antibiotic resistange gene profiles of the main species of ruminal bacteria and reveal the potential role of mobile genetic elements in shaping the resistome of the rumen microbiome, with implications for human and animal health.

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Complete Nucleotide Sequence of Two Multidrug-Resistant IncR Plasmids from Klebsiella pneumoniae

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02773-13 ◽

2014 ◽

Vol 58 (7) ◽

pp. 4207-4210 ◽

Cited By ~ 30

Author(s):

Fabrice Compain ◽

Lionel Frangeul ◽

Laurence Drieux ◽

Charlotte Verdet ◽

Sylvain Brisse ◽

...

Keyword(s):

Antibiotic Resistance ◽

Multidrug Resistance ◽

Nucleotide Sequence ◽

Resistance Genes ◽

Complete Nucleotide Sequence ◽

Clinical Importance ◽

Antibiotic Resistance Genes ◽

Multidrug Resistant ◽

Resistance Determinants ◽

Gene Carriers

ABSTRACTWe report here the complete nucleotide sequence of two IncR replicons encoding multidrug resistance determinants, including β-lactam (blaDHA-1,blaSHV-12), aminoglycoside (aphA1,strA,strB), and fluoroquinolone (qnrB4,aac6′-1b-cr) resistance genes. The plasmids have backbones that are similar to each other, including the replication and stability systems, and contain a wide variety of transposable elements carrying known antibiotic resistance genes. This study confirms the increasing clinical importance of IncR replicons as resistance gene carriers.

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Enterococcus faecalisCRISPR-Cas is a robust barrier to conjugative antibiotic resistance dissemination in the murine intestine

10.1101/312751 ◽

2018 ◽

Cited By ~ 2

Author(s):

Valerie J. Price ◽

Sara W. McBride ◽

Karthik Hullahalli ◽

Anushila Chatterjee ◽

Breck A. Duerkop ◽

...

Keyword(s):

Antibiotic Resistance ◽

Enterococcus Faecalis ◽

Resistance Genes ◽

Antibiotic Resistance Genes ◽

Conjugative Plasmids ◽

Resistance Plasmids ◽

The Impact ◽

Mammalian Intestine

AbstractCRISPR-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 inin vivomodels relevant to infectious disease are lacking. These are significant gaps in knowledge because conjugative plasmids disseminate antibiotic resistance genes among pathogensin 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 faecalisis a native colonizer of the mammalian intestine and harbors pheromone-responsive plasmids (PRPs). PRPs mediate inter- and intraspecies transfer of antibiotic resistance genes. We assessedE. faecalisCRISPR-Cas anti-PRP activity in the mouse intestine and under varyingin vitroconditions. We observed striking differences in CRISPR-Cas efficiencyin vitroversusin vivo. With few exceptions, CRISPR-Cas blocked intestinal PRP dissemination, whilein vitro, the PRP frequently escaped CRISPR-Cas defense. Our results further the understanding of CRISPR-Cas biology by demonstrating that standardin vitroexperiments do not adequately model thein vivoanti-plasmid activity of CRISPR-Cas. Additionally, our work identifies several variables that impact the apparentin vitroanti-plasmid activity of CRISPR-Cas, including planktonic versus biofilm settings, different donor/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 human microbiota can have significant impacts onin vivoantibiotic resistance dissemination.ImportanceCRISPR-Cas is a type of immune system encoded by 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 varyingin vitroconditions. We observed a robust effect of CRISPR-Cas onin vivobut notin vitrodissemination of antibiotic resistance plasmids in the native mammalian intestinal colonizerEnterococcus faecalis. We conclude that standard laboratory experiments currently do not appropriately model thein vivoconditions where antibiotic resistance dissemination occurs betweenE. faecalisstrains. Moreover, our results demonstrate that CRISPR-Cas encoded by native members of the mammalian intestinal microbiota can block the spread of antibiotic resistance plasmids.

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Screening and Quantification of the Expression of Antibiotic Resistance Genes in Acinetobacter baumannii with a Microarray

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01037-09 ◽

2009 ◽

Vol 54 (1) ◽

pp. 333-340 ◽

Cited By ~ 59

Author(s):

Sébastien Coyne ◽

Ghislaine Guigon ◽

Patrice Courvalin ◽

Bruno Périchon

Keyword(s):

Antibiotic Resistance ◽

Acinetobacter Baumannii ◽

Resistance Genes ◽

Efflux Pump ◽

Acquired Resistance ◽

Antibiotic Resistance Genes ◽

Efflux Pumps ◽

Single Step ◽

Resistance Determinants

ABSTRACT An oligonucleotide-based DNA microarray was developed to evaluate expression of genes for efflux pumps in Acinetobacter baumannii and to detect acquired antibiotic resistance determinants. The microarray contained probes for 205 genes, including those for 47 efflux systems, 55 resistance determinants, and 35 housekeeping genes. The microarray was validated by comparative analysis of mutants overexpressing or deficient in the pumps relative to the parental strain. The performance of the microarray was also evaluated using in vitro single-step mutants obtained on various antibiotics. Overexpression, confirmed by quantitative reverse transcriptase PCR, of RND efflux pumps AdeABC, due to a G30D substitution in AdeS in a multidrug-resistant (MDR) strain obtained on gentamicin, and AdeIJK, in two mutants obtained on cefotaxime or tetracycline, was detected. A new efflux pump, AdeFGH, was found to be overexpressed in a mutant obtained on chloramphenicol. Study of MDR clinical isolates, including the AYE strain, whose entire sequence has been determined, indicated overexpression of AdeABC and of the chromosomally encoded cephalosporinase as well as the presence of several acquired resistance genes. The overexpressed and acquired determinants detected by the microarray could account for nearly the entire MDR phenotype of the isolates. The microarray is potentially useful for detection of resistance in A. baumannii and should allow detection of new efflux systems associated with antibiotic resistance.

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Retail Ready-to-Eat Food as a Potential Vehicle for Staphylococcus spp. Harboring Antibiotic Resistance Genes

Journal of Food Protection ◽

10.4315/0362-028x.jfp-13-466 ◽

2014 ◽

Vol 77 (6) ◽

pp. 993-998 ◽

Cited By ~ 16

Author(s):

WIOLETA CHAJĘCKA-WIERZCHOWSKA ◽

ANNA ZADERNOWSKA ◽

BEATA NALEPA ◽

MAGDA SIERPI´NSKA ◽

ŁUCJA ŁANIEWSKA-TROKENHEIM

Keyword(s):

Antibiotic Resistance ◽

Resistance Genes ◽

Meca Gene ◽

Antibiotic Resistance Genes ◽

Tetracycline Resistance ◽

Multidrug Resistant ◽

Resistant Bacteria ◽

Antibiotic Resistant ◽

Cured Meats ◽

Staphylococcus Spp

Ready-to-eat (RTE) food, which does not need thermal processing before consumption, could be a vehicle for the spread of antibiotic-resistant microorganisms. As part of general microbiological safety checks, staphylococci are routinely enumerated in these kinds of foods. However, the presence of antibiotic-resistant staphylococci in RTE food is not routinely investigated, and data are only available from a small number of studies. The present study evaluated the pheno- and genotypical antimicrobial resistance profile of Staphylococcus spp. isolated from 858 RTE foods (cheeses, cured meats, sausages, smoked fishes, salads). Of 113 strains isolated, S. aureus was the most prevalent species, followed by S. xylosus, S. saprophyticus, and S. epidermidis. More than half (54.9%) of the isolates were resistant to at least one class of tested antibiotic; of these, 35.4% of the strains were classified as multidrug resistant. Most of the isolates were resistant to cefoxitin (49.6%), followed by clindamycin (39.3%), tigecycline (27.4%), quinupristin-dalfopristin (22.2%), rifampin (20.5%), tetracycline (17.9%), and erythromycin (8.5%). All methicillin-resistant staphylococci harbored the mecA gene. Among the isolates resistant to at least one antibiotic, 38 harbored tetracycline resistance determinant tet(M), 24 harbored tet(L), and 9 harbored tet(K). Of the isolates positive for tet(M) genes, 34.2% were positive for the Tn916-Tn1545–like integrase family gene. Our results indicated that retail RTE food could be considered an important route for the transmission of antibiotic-resistant bacteria harboring multiple antibiotic resistance genes.

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Design and Preclinical Development of a Phage Product for the Treatment of Antibiotic-Resistant Staphylococcus aureus Infections

Viruses ◽

10.3390/v11010088 ◽

2019 ◽

Vol 11 (1) ◽

pp. 88 ◽

Cited By ~ 40

Author(s):

Susan Lehman ◽

Gillian Mearns ◽

Deborah Rankin ◽

Robert Cole ◽

Frenk Smrekar ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Antibiotic Resistance ◽

Phase 1 ◽

Antibiotic Resistance Genes ◽

Multidrug Resistant ◽

Preclinical Development ◽

Antibiotic Resistant ◽

Fixed Composition ◽

Spontaneous Frequency

Bacteriophages, viruses that only kill specific bacteria, are receiving substantial attention as nontraditional antibacterial agents that may help alleviate the growing antibiotic resistance problem in medicine. We describe the design and preclinical development of AB-SA01, a fixed-composition bacteriophage product intended to treat Staphylococcus aureus infections. AB-SA01 contains three naturally occurring, obligately lytic myoviruses related to Staphylococcus phage K. AB-SA01 component phages have been sequenced and contain no identifiable bacterial virulence or antibiotic resistance genes. In vitro, AB-SA01 killed 94.5% of 401 clinical Staphylococcus aureus isolates, including methicillin-resistant and vancomycin-intermediate ones for a total of 95% of the 205 known multidrug-resistant isolates. The spontaneous frequency of resistance to AB-SA01 was ≤3 × 10−9, and resistance emerging to one component phage could be complemented by the activity of another component phage. In both neutropenic and immunocompetent mouse models of acute pneumonia, AB-SA01 reduced lung S. aureus populations equivalently to vancomycin. Overall, the inherent characteristics of AB-SA01 component phages meet regulatory and generally accepted criteria for human use, and the preclinical data presented here have supported production under good manufacturing practices and phase 1 clinical studies with AB-SA01.

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