Multiplex CRISPRi-Cas9 silencing of planktonic and stage-specific biofilm genes in Enterococcus faecalis

ABSTRACTEnterococcus faecalis is an opportunistic pathogen, which can cause multidrug-resistant life-threatening infections. Gaining a complete understanding of enterococcal pathogenesis is a crucial step in identifying a strategy to effectively treat enterococcal infections. However, bacterial pathogenesis is a complex process often involving a combination of genes and multi-level regulation. Compared to established knockout methodologies, CRISPRi approaches enable rapid and efficient silencing of genes to interrogate gene products and pathways involved in pathogenesis. As opposed to traditional gene inactivation approaches, CRISPRi can also be quickly repurposed for multiplexing or used to study essential genes. Here we have developed a novel dual-vector nisin-inducible CRISPRi system in E. faecalis that can efficiently silence via both non-template and template strand targeting. Since nisin-controlled gene expression system is functional in various Gram-positive bacteria, the developed CRISPRi tool can be extended to other genera. This system can be applied to study essential genes, genes involved in antimicrobial resistance, and genes involved in biofilm formation and persistence. The system is robust, and can be scaled up for high-throughput screens or combinatorial targeting. This tool substantially enhances our ability to study enterococcal biology and pathogenesis, host-bacteria interactions, and inter-species communication.IMPORTANCEEnterococcus faecalis causes multidrug resistant life-threatening infections, and is often co-isolated with other pathogenic bacteria from polymicrobial biofilm-associated infections. Genetic tools to dissect complex interactions in mixed microbial communities are largely limited to transposon mutagenesis and traditional time- and labour-intensive allelic exchange methods. Built upon streptococcal dCas9, we developed an easily-modifiable, inducible CRISPRi system for E. faecalis that can efficiently silence single and multiple genes. This system can silence genes involved in biofilm formation, antibiotic resistance, and can be used to interrogate gene essentiality. Uniquely, this tool is optimized to study genes important for biofilm initiation, maturation, and maintenance, and can be used to perturb pre-formed biofilms. This system will be valuable to rapidly and efficiently investigate a wide range of aspects of complex enterococcal biology.

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Multiplex CRISPRi System Enables the Study of Stage-Specific Biofilm Genetic Requirements in Enterococcus faecalis

mBio ◽

10.1128/mbio.01101-20 ◽

2020 ◽

Vol 11 (5) ◽

Cited By ~ 2

Author(s):

Irina Afonina ◽

June Ong ◽

Jerome Chua ◽

Timothy Lu ◽

Kimberly A. Kline

Keyword(s):

Enterococcus Faecalis ◽

Biofilm Formation ◽

Pathogenic Bacteria ◽

Expression System ◽

Multidrug Resistant ◽

Essential Genes ◽

Host Bacterium ◽

Content Type ◽

Wide Range ◽

Life Threatening

ABSTRACT Enterococcus faecalis is an opportunistic pathogen, which can cause multidrug-resistant life-threatening infections. Gaining a complete understanding of enterococcal pathogenesis is a crucial step in identifying a strategy to effectively treat enterococcal infections. However, bacterial pathogenesis is a complex process often involving a combination of genes and multilevel regulation. Compared to established knockout methodologies, CRISPR interference (CRISPRi) approaches enable the rapid and efficient silencing of genes to interrogate gene products and pathways involved in pathogenesis. As opposed to traditional gene inactivation approaches, CRISPRi can also be quickly repurposed for multiplexing or used to study essential genes. Here, we have developed a novel dual-vector nisin-inducible CRISPRi system in E. faecalis that can efficiently silence via both nontemplate and template strand targeting. Since the nisin-controlled gene expression system is functional in various Gram-positive bacteria, the developed CRISPRi tool can be extended to other genera. This system can be applied to study essential genes, genes involved in antimicrobial resistance, and genes involved in biofilm formation and persistence. The system is robust and can be scaled up for high-throughput screens or combinatorial targeting. This tool substantially enhances our ability to study enterococcal biology and pathogenesis, host-bacterium interactions, and interspecies communication. IMPORTANCE Enterococcus faecalis causes multidrug-resistant life-threatening infections and is often coisolated with other pathogenic bacteria from polymicrobial biofilm-associated infections. Genetic tools to dissect complex interactions in mixed microbial communities are largely limited to transposon mutagenesis and traditional time- and labor-intensive allelic-exchange methods. Built upon streptococcal dCas9, we developed an easily modifiable, inducible CRISPRi system for E. faecalis that can efficiently silence single and multiple genes. This system can silence genes involved in biofilm formation and antibiotic resistance and can be used to interrogate gene essentiality. Uniquely, this tool is optimized to study genes important for biofilm initiation, maturation, and maintenance and can be used to perturb preformed biofilms. This system will be valuable to rapidly and efficiently investigate a wide range of aspects of complex enterococcal biology.

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CRISPRi enables studies of enterococcal biofilm initiation, maturation and maintenance.

10.5194/biofilms9-40 ◽

2020 ◽

Author(s):

Irina Afonina ◽

Timothy Lu ◽

Kimberly Kline

Keyword(s):

Patient Outcomes ◽

Biofilm Formation ◽

Pathogenic Bacteria ◽

Multidrug Resistant ◽

Allelic Exchange ◽

Complex Interactions ◽

Interference System ◽

Wide Range ◽

Life Threatening ◽

Human Infections

Among the Enterococci, Enterococcus faecalis is most frequently associated with human infections ranging from the urinary tract and wound infection to endocarditis and bacteraemia. These infections are often multidrug-resistant and, hence, life-threatening. Moreover, E. faecalis are often co-isolated with other pathogenic bacteria from polymicrobial biofilm-associated infections contributing to disease progression and poorer patient outcomes. Genetic tools to dissect complex interactions in biofilms and mixed microbial communities are largely limited to transposon mutagenesis and traditional allelic exchange methods requiring time- and labour-intensive two-step integration and excision screening that can take a week or more to make a single mutant. We built upon the well-characterized CRISPR interference system using streptococcal dCas9 to develop an easily-modifiable, inducible system for E. faecalis that can efficiently silence single and multiple genes in a matter of hours. We show that this system can silence genes involved in biofilm formation, antibiotic resistance, and can be used to interrogate gene essentiality. Uniquely, this tool is optimized to study genes important for biofilm initiation, maturation, and maintenance, and can be used to perturb pre-formed biofilms. This inducible CRISPRi system will be valuable to rapidly and efficiently investigate a wide range of aspects of complex enterococcal regulation networks within the biofilms, including polymicrobial biofilms.&#160;

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Interactions between Candida albicans and Enterococcus faecalis in an Organotypic Oral Epithelial Model

Microorganisms ◽

10.3390/microorganisms8111771 ◽

2020 ◽

Vol 8 (11) ◽

pp. 1771

Author(s):

Akshaya Lakshmi Krishnamoorthy ◽

Alex A. Lemus ◽

Adline Princy Solomon ◽

Alex M. Valm ◽

Prasanna Neelakantan

Keyword(s):

Candida Albicans ◽

Enterococcus Faecalis ◽

Biofilm Formation ◽

Opportunistic Pathogen ◽

Surface Erosion ◽

Host Immune System ◽

Microbial Invasion ◽

Mucosal Surfaces ◽

Life Threatening ◽

Hyphal Formation

Candida albicans as an opportunistic pathogen exploits the host immune system and causes a variety of life-threatening infections. The polymorphic nature of this fungus gives it tremendous advantage to breach mucosal barriers and cause oral and disseminated infections. Similar to C. albicans, Enterococcus faecalis is a major opportunistic pathogen, which is of critical concern in immunocompromised patients. There is increasing evidence that E. faecalis co-exists with C. albicans in the human body in disease samples. While the interactive profiles between these two organisms have been studied on abiotic substrates and mouse models, studies on their interactions on human oral mucosal surfaces are non-existent. Here, for the first time, we comprehensively characterized the interactive profiles between laboratory and clinical isolates of C. albicans (SC5314 and BF1) and E. faecalis (OG1RF and P52S) on an organotypic oral mucosal model. Our results demonstrated that the dual species biofilms resulted in profound surface erosion and significantly increased microbial invasion into mucosal compartments, compared to either species alone. Notably, several genes of C. albicans involved in tissue adhesion, hyphal formation, fungal invasion, and biofilm formation were significantly upregulated in the presence of E. faecalis. By contrast, E. faecalis genes involved in quorum sensing, biofilm formation, virulence, and mammalian cell invasion were downregulated. This study highlights the synergistic cross-kingdom interactions between E. faecalis and C. albicans in mucosal tissue invasion.

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Comparative biofilm assays using Enterococcus faecalis OG1RF identify new determinants of biofilm formation

10.1101/2021.02.24.432758 ◽

2021 ◽

Author(s):

Julia L. E. Willett ◽

Jennifer L. Dale ◽

Lucy M. Kwiatkowski ◽

Jennifer L. Powers ◽

Michelle L. Korir ◽

...

Keyword(s):

Enterococcus Faecalis ◽

Growth Medium ◽

Biofilm Formation ◽

Time Course ◽

Opportunistic Pathogen ◽

Genetic Determinants ◽

Experimental Conditions ◽

Biofilm Reactors ◽

Biofilm Architecture ◽

Biofilm Development

AbstractEnterococcus faecalis is a common commensal organism and a prolific nosocomial pathogen that causes biofilm-associated infections. Numerous E. faecalis OG1RF genes required for biofilm formation have been identified, but few studies have compared genetic determinants of biofilm formation and biofilm morphology across multiple conditions. Here, we cultured transposon (Tn) libraries in CDC biofilm reactors in two different media and used Tn sequencing (TnSeq) to identify core and accessory biofilm determinants, including many genes that are poorly characterized or annotated as hypothetical. Multiple secondary assays (96-well plates, submerged Aclar, and MultiRep biofilm reactors) were used to validate phenotypes of new biofilm determinants. We quantified biofilm cells and used fluorescence microscopy to visualize biofilms formed by 6 Tn mutants identified using TnSeq and found that disrupting these genes (OG1RF_10350, prsA, tig, OG1RF_10576, OG1RF_11288, and OG1RF_11456) leads to significant time- and medium-dependent changes in biofilm architecture. Structural predictions revealed potential roles in cell wall homeostasis for OG1RF_10350 and OG1RF_11288 and signaling for OG1RF_11456. Additionally, we identified growth medium-specific hallmarks of OG1RF biofilm morphology. This study demonstrates how E. faecalis biofilm architecture is modulated by growth medium and experimental conditions, and identifies multiple new genetic determinants of biofilm formation.ImportanceE. faecalis is an opportunistic pathogen and a leading cause of hospital-acquired infections, in part due to its ability to form biofilms. A complete understanding of the genes required for E. faecalis biofilm formation as well as specific features of biofilm morphology related to nutrient availability and growth conditions is crucial for understanding how E. faecalis biofilm-associated infections develop and resist treatment in patients. We employed a comprehensive approach to analysis of biofilm determinants by combining TnSeq primary screens with secondary phenotypic validation using diverse biofilm assays. This enabled identification of numerous core (important under many conditions) and accessory (important under specific conditions) biofilm determinants in E. faecalis OG1RF. We found multiple genes whose disruption results in drastic changes to OG1RF biofilm morphology. These results expand our understanding of the genetic requirements for biofilm formation in E. faecalis that affect the time course of biofilm development as well as the response to specific nutritional conditions.

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F-type Pyocins are Diverse Non-Contractile Phage Tail-Like Weapons for Killing Pseudomonas aeruginosa

10.1101/2021.02.16.431561 ◽

2021 ◽

Author(s):

Senjuti Saha ◽

Chidozie D. Ojobor ◽

Erik Mackinnon ◽

Olesia I. North ◽

Joseph Bondy-Denomy ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Bactericidal Activity ◽

Pathogenic Bacteria ◽

Therapeutic Potential ◽

Experimental Studies ◽

Opportunistic Pathogen ◽

Multidrug Resistant ◽

Antibiotic Resistant ◽

Genes Encoding ◽

Intraspecies Competition

ABSTRACTMost Pseudomonas aeruginosa strains produce bacteriocins derived from contractile or non-contractile phage tails known as R-type and F-type pyocins, respectively. These bacteriocins possess strain-specific bactericidal activity against P. aeruginosa and likely increase evolutionary fitness through intraspecies competition. R-type pyocins have been studied extensively and show promise as alternatives to antibiotics. Although they have similar therapeutic potential, experimental studies on F-type pyocins are limited. Here, we provide a bioinformatic and experimental investigation of F-type pyocins. We introduce a systematic naming scheme for genes found in R- and F-type pyocin operons and identify 15 genes invariably found in strains producing F-type pyocins. Five proteins encoded at the 3’-end of the F-type pyocin cluster are divergent in sequence, and likely determine bactericidal specificity. We use sequence similarities among these proteins to define 11 distinct F-type pyocin groups, five of which had not been previously described. The five genes encoding the variable proteins associate in two modules that have clearly re-assorted independently during the evolution of these operons. These proteins are considerably more diverse than the specificity-determining tail fibers of R-type pyocins, suggesting that F-type pyocins emerged earlier or have been subject to distinct evolutionary pressures. Experimental studies on six F-type pyocin groups show that each displays a distinct spectrum of bactericidal activity. This activity is strongly influenced by the lipopolysaccharide O-antigen type, but other factors also play a role. F-type pyocins appear to kill as efficiently as R-type pyocins. These studies set the stage for the development of F-type pyocins as anti-bacterial therapeutics.IMPORTANCEPseudomonas aeruginosa is an opportunistic pathogen that causes a broad spectrum of antibiotic resistant infections with high mortality rates, particularly in immunocompromised individuals and cystic fibrosis patients. Due to the increasing frequency of multidrug-resistant P. aeruginosa infections, there is great interest in the development of alternative therapeutics. One alternative is protein-based antimicrobials called bacteriocins, which are produced by one strain of bacteria to kill other strains. In this study, we investigate F-type pyocins, bacteriocins naturally produced by P. aeruginosa that resemble non-contractile phage tails. We show that they are potent killers of P. aeruginosa, and distinct pyocin groups display different killing specificities. We have identified the probable specificity determinants of F-type pyocins, which opens up the potential to engineer them to precisely target strains of pathogenic bacteria. The resemblance of F-type pyocins to well characterized phage tails will greatly facilitate their development into effective antibacterials.

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Biofilm Formation and Detection of Fluoroquinolone- and Carbapenem-Resistant Genes in Multidrug-Resistant Acinetobacter baumannii

Canadian Journal of Infectious Diseases and Medical Microbiology ◽

10.1155/2019/3454907 ◽

2019 ◽

Vol 2019 ◽

pp. 1-5 ◽

Cited By ~ 7

Author(s):

María-Guadalupe Avila-Novoa ◽

Oscar-Alberto Solís-Velázquez ◽

Daniel-Eduardo Rangel-López ◽

Jean-Pierre González-Gómez ◽

Pedro-Javier Guerrero-Medina ◽

...

Keyword(s):

Acinetobacter Baumannii ◽

Biofilm Formation ◽

Opportunistic Pathogen ◽

Multidrug Resistant ◽

Resistance Mechanisms ◽

Low Grade ◽

Carbapenem Resistant ◽

Hospital Environments ◽

Potential Association ◽

Clinical Environments

Acinetobacter baumannii is an important opportunistic pathogen that shows resistance to cephalosporins, penicillins, carbapenems, fluoroquinolones, and aminoglycosides, the multiresistance being associated with its ability to form biofilms in clinical environments. The aim of this study was to determine biofilm formation and its potential association with genes involved in antibiotic resistance mechanisms of A. baumannii isolates of different clinical specimens. We demonstrated 100% of the A. baumannii isolates examined to be multidrug resistant (MDR), presenting a 73.3% susceptibility to cefepime and a 53.3% susceptibility to ciprofloxacin. All A. baumannii isolates were positive for blaOXA-51, 33.3% being positive for blaOXA-23 and ISAba1, and 73.3% being positive for gyrA. We found 86.6% of A. baumannii strains to be low-grade biofilm formers and 13.3% to be biofilm negative; culturing on Congo red agar (CRA) plates revealed that 73.3% of the A. baumannii isolates to be biofilm producers, while 26.6% were not. These properties, combined with the role of A. baumannii as a nosocomial pathogen, increase the probability of A. baumannii causing nosocomial infections and outbreaks as a complication during therapeutic treatments and emphasize the need to control A. baumannii biofilms in hospital environments.

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In Vitro and In Silico Evaluation of Biological Activity of a New Series of Oxadiazole Compounds Against Esp Gene Expression in Enterococcus faecalis Biofilm

Gene Cell and Tissue ◽

10.5812/gct.112403 ◽

2021 ◽

Vol In Press (In Press) ◽

Author(s):

Sepideh Ghameshlouei ◽

Nakisa Zarrabi Ahrabi ◽

Yasin SarveAhrabi

Keyword(s):

Enterococcus Faecalis ◽

Biofilm Formation ◽

Biological Activities ◽

Control Sample ◽

Surface Protein ◽

Bacterial Biofilm ◽

Receptor Binding Site ◽

Wide Range ◽

Oxadiazole Derivatives ◽

The Impact

Background: The enterococcal surface protein (Esp) is a high-molecular-weight surface protein of biofilm creating agent in Enterococcus faecalis. Oxadiazoles have a wide range of biological activities. Objective: This research aimed to examine the impact of new oxadiazole derivatives on the expression of Esp, playing an important role in promoting the biofilm formation ability of drug-resistant E. faecalis strains. Method: 1, 3, 4-oxadiazole derivatives were synthesized through a one-step synthesis. E. faecalis strains were collected and isolated from hospitals in Tehran. The antimicrobial properties of the synthesized materials against the isolated strains were investigated. RNA, DNA, and cDNA were extracted, and the relative expression of Esp in E. faecalis isolates was evaluated by real-time PCR. Docking study was performed by AutoDock vina software, and the resulting docking poses were analyzed using Discovery Studio 4.5 Client software. Results: The use of synthesized derivatives changed the Esp expression level in different isolates compared to the control sample. The two compounds containing naphthalene (4f) and methoxyphenyl (4g) caused respectively a 2-fold and a 3-fold decrease in Esp expression compared to the control sample. The compound 4f with the best binding energy among the compounds (-9.2) had the most hydrogen and hydrophobic bonds with the receptor-binding site. Conclusions: 1, 3, 4-oxadiazole derivatives, especially naphthalene and methoxyphenyl, act as inhibitors of bacterial biofilm formation and can be used in the pharmaceutical and biological industries.

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Inhibition of Biofilm Formation by the Synergistic Action of EGCG-S and Antibiotics

Antibiotics ◽

10.3390/antibiotics10020102 ◽

2021 ◽

Vol 10 (2) ◽

pp. 102

Author(s):

Shrameeta Shinde ◽

Lee H. Lee ◽

Tinchun Chu

Keyword(s):

Green Tea ◽

Biofilm Formation ◽

Pathogenic Bacteria ◽

Physiological State ◽

Epigallocatechin Gallate ◽

Multidrug Resistant ◽

Mortality And Morbidity ◽

Viability Assay ◽

Green Tea Polyphenol ◽

Synergistic Agent

Biofilm, a stress-induced physiological state, is an established means of antimicrobial tolerance. A perpetual increase in multidrug resistant (MDR) infections associated with high mortality and morbidity have been observed in healthcare settings. Multiple studies have indicated that the use of natural products can prevent bacterial growth. Recent studies in the field have identified that epigallocatechin gallate (EGCG), a green tea polyphenol, could disrupt bacterial biofilms. A modified lipid-soluble EGCG, epigallocatechin-3-gallate-stearate (EGCG-S), has enhanced the beneficial properties of green tea. This study focuses on utilizing EGCG-S as a novel synergistic agent with antibiotics to prevent or control biofilm. Different formulations of EGCG-S and selected antibiotics were used to study their combinatorial effects on biofilms produced by five potential pathogenic bacteria, Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Staphylococcusepidermidis, and Mycobacterium smegmatis. The crystal violet (CV) assay and the sensitive fluorescence-based resazurin biofilm viability assay were used to assess the biofilm production. Our results identified optimal formulation for each bacterium, effectively inhibiting biofilm formation to an extent of 95–99%. Colony-forming unit (CFU) and cell viability analyses showed a decrease of viable bacteria. These results depict the potential of EGCG-S as a synergistic agent with antibiotics and as an anti-biofilm agent.

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Enterococcus faecalis and Vibrio harveyicolonize low-density polyethylene and biodegradable plastics under marine conditions

FEMS Microbiology Letters ◽

10.1093/femsle/fnaa125 ◽

2020 ◽

Vol 367 (15) ◽

Cited By ~ 1

Author(s):

Ilef Hchaichi ◽

Francesca Bandini ◽

Giulia Spini ◽

Mohamed Banni ◽

Pier Sandro Cocconcelli ◽

...

Keyword(s):

Enterococcus Faecalis ◽

Biofilm Formation ◽

Aquatic Ecosystems ◽

Pathogenic Bacteria ◽

Sea Water ◽

Vibrio Harveyi ◽

Low Density Polyethylene ◽

Low Density ◽

Plastic Surfaces ◽

Scanning Electron

ABSTRACT Conventional plastics and bioplastics are known to accumulate in aquatic ecosystems, emerging as new surfaces for biofilm formation and gene exchanges. On the other hand, the fate of non-conventional bioplastics in the marine environment is still unclear. In this study we have measured, by means of crystal violet test and scanning electron microscopy (SEM), the ability of two model bacteria, Vibrio harveyi and Enterococcus faecalis, to form biofilms on low-density polyethylene (PE), polylactic acid (PLA) and starch-based bioplastic (SBB) surfaces. Experiments were conducted in artificial sea water, incubating squares of 3 cm2 of the three plastics with the two model microorganisms and sampling overnight, and at 3 and 6 days. The presence of biofilms on plastic surfaces was detected from day one of incubation and SBB was the material with the highest biofilm formation. E. faecalis formed the thickest biofilm after 3 days with PLA and SBB, but did not remain stable, and after 6 days with PE. The maximum biofilm formation of V. harveyi was obtained overnight with SBB and PE, and after 3 days with PLA. Our data indicate that both plastic and bioplastics support the formation of biofilms of model pathogenic bacteria, highlighting potential concerns for human health.

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Thiostrepton Hijacks Pyoverdine Receptors To Inhibit Growth ofPseudomonas aeruginosa

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00472-19 ◽

2019 ◽

Vol 63 (9) ◽

Cited By ~ 8

Author(s):

Michael R. M. Ranieri ◽

Derek C. K. Chan ◽

Luke N. Yaeger ◽

Madeleine Rudolph ◽

Sawyer Karabelas-Pittman ◽

...

Keyword(s):

Biofilm Formation ◽

Opportunistic Pathogen ◽

Multidrug Resistant ◽

Clinical Isolates ◽

23S Rrna ◽

Dependent Manner ◽

Peptide Antibiotics ◽

Iron Starvation ◽

Gram Negative ◽

Content Type

ABSTRACTPseudomonas aeruginosais a biofilm-forming opportunistic pathogen and is intrinsically resistant to many antibiotics. In a high-throughput screen for molecules that modulate biofilm formation, we discovered that the thiopeptide antibiotic thiostrepton (TS), which is considered to be inactive against Gram-negative bacteria, stimulatedP. aeruginosabiofilm formation in a dose-dependent manner. This phenotype is characteristic of exposure to antimicrobial compounds at subinhibitory concentrations, suggesting that TS was active againstP. aeruginosa. Supporting this observation, TS inhibited the growth of a panel of 96 multidrug-resistant (MDR)P. aeruginosaclinical isolates at low-micromolar concentrations. TS also had activity againstAcinetobacter baumanniiclinical isolates. The expression of Tsr, a 23S rRNA-modifying methyltransferase from TS producerStreptomyces azureus, intransconferred TS resistance, confirming that the drug acted via its canonical mode of action, inhibition of ribosome function. The deletion of oligopeptide permease systems used by other peptide antibiotics for uptake failed to confer TS resistance. TS susceptibility was inversely proportional to iron availability, suggesting that TS exploits uptake pathways whose expression is increased under iron starvation. Consistent with this finding, TS activity againstP. aeruginosaandA. baumanniiwas potentiated by the FDA-approved iron chelators deferiprone and deferasirox and by heat-inactivated serum. Screening ofP. aeruginosamutants for TS resistance revealed that it exploits pyoverdine receptors FpvA and FpvB to cross the outer membrane. We show that the biofilm stimulation phenotype can reveal cryptic subinhibitory antibiotic activity, and that TS has activity against select multidrug-resistant Gram-negative pathogens under iron-limited growth conditions, similar to those encountered at sites of infection.

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