scholarly journals Identification of Novel Bacteriophages with Therapeutic Potential That TargetEnterococcus faecalis

2019 ◽  
Vol 87 (11) ◽  
Author(s):  
M. Al-Zubidi ◽  
M. Widziolek ◽  
E. K. Court ◽  
A. F. Gains ◽  
R. E. Smith ◽  
...  
Keyword(s):  
Host Range ◽  
Root Canal ◽  
Therapeutic Potential ◽  
Opportunistic Pathogen ◽  
Cross Sectional ◽  
Antibiotic Resistant ◽  
Content Type ◽  
Endodontic Infection ◽  
Tail Protein

ABSTRACTThe Gram-positive opportunistic pathogenEnterococcus faecalisis frequently responsible for nosocomial infections in humans and represents one of the most common bacteria isolated from recalcitrant endodontic (root canal) infections.E. faecalisis intrinsically resistant to several antibiotics routinely used in clinical settings (such as cephalosporins and aminoglycosides) and can acquire resistance to vancomycin (vancomycin-resistant enterococci). The resistance ofE. faecalisto several classes of antibiotics and its capacity to form biofilms cause serious therapeutic problems. Here, we report the isolation of several bacteriophages that targetE. faecalisstrains isolated from the oral cavity of patients suffering root canal infections. All phages isolated wereSiphoviridaewith similar tail lengths (200 to 250 nm) and icosahedral heads. The genome sequences of three isolated phages were highly conserved with the exception of predicted tail protein genes that diverge in sequence, potentially reflecting the host range. The properties of the phage with the broadest host range (SHEF2) were further characterized. We show that this phage requires interaction with components of the major and variant region enterococcal polysaccharide antigen to engage in lytic infection. Finally, we explored the therapeutic potential of this phage and show that it can eradicateE. faecalisbiofilms formedin vitroon a standard polystyrene surface but also on a cross-sectional tooth slice model of endodontic infection. We also show that SHEF2 cleared a lethal infection of zebrafish when applied in the circulation. We therefore propose that the phage described here could be used to treat a broad range of antibiotic-resistantE. faecalisinfections.

scholarly journals Successful Development of Bacteriocins into Therapeutic Formulation for Treatment of MRSA Skin Infection in a Murine Model

2020 ◽  
Vol 64 (12) ◽  
Author(s):  
Kirill V. Ovchinnikov ◽  
Christian Kranjec ◽  
Tage Thorstensen ◽  
Harald Carlsen ◽  
Dzung B. Diep
Keyword(s):  
Skin Infection ◽  
Therapeutic Potential ◽  
Multidrug Resistant ◽  
Penicillin G ◽  
Infection Model ◽  
Component Mixture ◽  
Antibiotic Resistant ◽  
Content Type ◽  
Infection Treatment

ABSTRACT The emergence of antibiotic-resistant pathogens has caused a serious worldwide problem in infection treatment in recent years. One of the pathogens is methicillin-resistant Staphylococcus aureus (MRSA), which is a major cause of skin and soft tissue infections. Alternative strategies and novel sources of antimicrobials to solve antibiotic resistance problems are urgently needed. In this study, we explored the potential of two broad-spectrum bacteriocins, garvicin KS and micrococcin P1, in skin infection treatments. The two bacteriocins acted synergistically with each other and with penicillin G in killing MRSA in vitro. The MICs of the antimicrobials in the three-component mixture were 40 ng/ml for micrococcin P1 and 2 μg/ml for garvicin KS and penicillin G, which were 62, 16, and at least 1,250 times lower than their MICs when assessed individually. To assess its therapeutic potential further, we challenged the three-component formulation in a murine skin infection model with the multidrug-resistant luciferase-tagged MRSA Xen31, a strain derived from the clinical isolate S. aureus ATCC 33591. Using the tagged-luciferase activity as a reporter for the presence of Xen31 in wounds, we demonstrated that the three-component formulation was efficient in eradicating the pathogen from treated wounds. Furthermore, compared to Fucidin cream, which is an antibiotic commonly used in skin infection treatments, our formulation was also superior in terms of preventing resistance development.

scholarly journals Loss-of-Function Mutations inepaRConfer Resistance to ϕNPV1 Infection inEnterococcus faecalisOG1RF

2018 ◽  
Vol 62 (10) ◽  
Author(s):  
Khang Ho ◽  
Wenwen Huo ◽  
Savannah Pas ◽  
Ryan Dao ◽  
Kelli L. Palmer
Keyword(s):  
High Frequency ◽  
Opportunistic Pathogen ◽  
Loss Of Function ◽  
Genetic Determinants ◽  
Human Gastrointestinal Tract ◽  
Antibiotic Resistant ◽  
Content Type ◽  
Risk Patients ◽  
Extracellular Polymer

ABSTRACTEnterococcus faecalisis a Gram-positive opportunistic pathogen that inhabits the human gastrointestinal tract. Because of the high frequency of antibiotic resistance amongEnterococcusclinical isolates, interest in using phage to treat enterococcal infections and to decolonize high-risk patients for antibiotic-resistantEnterococcusis rising. Bacteria can evolve phage resistance, but there is little published information on these mechanisms inE. faecalis. In this report, we identified genetic determinants ofE. faecalisresistance to phage NPV1 (ϕNPV1). We found that loss-of-function mutations inepaRconfer ϕNPV1 resistance by blocking phage adsorption. We attribute the inability of the phage to adsorb to the modification or loss of an extracellular polymer in strains with inactivatedepaR. Phage-resistantepaRmutants exhibited increased daptomycin and osmotic stress susceptibilities. Our results demonstrate thatin vitrospontaneous resistance to ϕNPV1 comes at a cost inE. faecalisOG1RF.

scholarly journals ZN148 Is a Modular Synthetic Metallo-β-Lactamase Inhibitor That Reverses Carbapenem Resistance in Gram-Negative Pathogens In Vivo

2020 ◽  
Vol 64 (6) ◽  
Author(s):  
Ørjan Samuelsen ◽  
Ove Alexander Høgmoen Åstrand ◽  
Christopher Fröhlich ◽  
Adam Heikal ◽  
Susann Skagseth ◽  
...  
Keyword(s):  
Active Site ◽  
Therapeutic Potential ◽  
Treatment Options ◽  
Carbapenem Resistance ◽  
Functional Space ◽  
Bactericidal Effect ◽  
Gram Negative ◽  
Content Type

ABSTRACT Carbapenem-resistant Gram-negative pathogens are a critical public health threat and there is an urgent need for new treatments. Carbapenemases (β-lactamases able to inactivate carbapenems) have been identified in both serine β-lactamase (SBL) and metallo-β-lactamase (MBL) families. The recent introduction of SBL carbapenemase inhibitors has provided alternative therapeutic options. Unfortunately, there are no approved inhibitors of MBL-mediated carbapenem-resistance and treatment options for infections caused by MBL-producing Gram-negatives are limited. Here, we present ZN148, a zinc-chelating MBL-inhibitor capable of restoring the bactericidal effect of meropenem and in vitro clinical susceptibility to carbapenems in >98% of a large international collection of MBL-producing clinical Enterobacterales strains (n = 234). Moreover, ZN148 was able to potentiate the effect of meropenem against NDM-1-producing Klebsiella pneumoniae in a murine neutropenic peritonitis model. ZN148 showed no inhibition of the human zinc-containing enzyme glyoxylase II at 500 μM, and no acute toxicity was observed in an in vivo mouse model with cumulative dosages up to 128 mg/kg. Biochemical analysis showed a time-dependent inhibition of MBLs by ZN148 and removal of zinc ions from the active site. Addition of exogenous zinc after ZN148 exposure only restored MBL activity by ∼30%, suggesting an irreversible mechanism of inhibition. Mass-spectrometry and molecular modeling indicated potential oxidation of the active site Cys221 residue. Overall, these results demonstrate the therapeutic potential of a ZN148-carbapenem combination against MBL-producing Gram-negative pathogens and that ZN148 is a highly promising MBL inhibitor that is capable of operating in a functional space not presently filled by any clinically approved compound.

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

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.

scholarly journals Rifabutin Is Bactericidal against Intracellular and Extracellular Forms of Mycobacterium abscessus

2020 ◽  
Vol 64 (11) ◽  
Author(s):  
Matt D. Johansen ◽  
Wassim Daher ◽  
Françoise Roquet-Banères ◽  
Clément Raynaud ◽  
Matthéo Alcaraz ◽  
...  
Keyword(s):  
Opportunistic Pathogen ◽  
Mycobacterium Abscessus ◽  
Zebrafish Model ◽  
Content Type ◽  
Attractive Option ◽  
Conventional Antibiotics ◽  
Susceptibility Profiles ◽  
Combination Regimens

ABSTRACT Mycobacterium abscessus is increasingly recognized as an emerging opportunistic pathogen causing severe lung diseases. As it is intrinsically resistant to most conventional antibiotics, there is an unmet medical need for effective treatments. Repurposing of clinically validated pharmaceuticals represents an attractive option for the development of chemotherapeutic alternatives against M. abscessus infections. In this context, rifabutin (RFB) has been shown to be active against M. abscessus and has raised renewed interest in using rifamycins for the treatment of M. abscessus pulmonary diseases. Here, we compared the in vitro and in vivo activity of RFB against the smooth and rough variants of M. abscessus, differing in their susceptibility profiles to several drugs and physiopathologial characteristics. While the activity of RFB is greater against rough strains than in smooth strains in vitro, suggesting a role of the glycopeptidolipid layer in susceptibility to RFB, both variants were equally susceptible to RFB inside human macrophages. RFB treatment also led to a reduction in the number and size of intracellular and extracellular mycobacterial cords. Furthermore, RFB was highly effective in a zebrafish model of infection and protected the infected larvae from M. abscessus-induced killing. This was corroborated by a significant reduction in the overall bacterial burden, as well as decreased numbers of abscesses and cords, two major pathophysiological traits in infected zebrafish. This study indicates that RFB is active against M. abscessus both in vitro and in vivo, further supporting its potential usefulness as part of combination regimens targeting this difficult-to-treat mycobacterium.

scholarly journals Mutant and Recombinant Phages Selected from In Vitro Coevolution Conditions Overcome Phage-Resistant Listeria monocytogenes

2020 ◽  
Vol 86 (22) ◽  
Author(s):  
Tracey Lee Peters ◽  
Yaxiong Song ◽  
Daniel W. Bryan ◽  
Lauren K. Hudson ◽  
Thomas G. Denes
Keyword(s):  
Listeria Monocytogenes ◽  
Host Range ◽  
Foodborne Pathogen ◽  
Resistant Bacteria ◽  
Phage Resistance ◽  
Short Term ◽  
Content Type ◽  
Life Threatening ◽  
The Impact

ABSTRACT Bacteriophages (phages) are currently available for use by the food industry to control the foodborne pathogen Listeria monocytogenes. Although phage biocontrols are effective under specific conditions, their use can select for phage-resistant bacteria that repopulate phage-treated environments. Here, we performed short-term coevolution experiments to investigate the impact of single phages and a two-phage cocktail on the regrowth of phage-resistant L. monocytogenes and the adaptation of the phages to overcome this resistance. We used whole-genome sequencing to identify mutations in the target host that confer phage resistance and in the phages that alter host range. We found that infections with Listeria phages LP-048, LP-125, or a combination of both select for different populations of phage-resistant L. monocytogenes bacteria with different regrowth times. Phages isolated from the end of the coevolution experiments were found to have gained the ability to infect phage-resistant mutants of L. monocytogenes and L. monocytogenes strains previously found to be broadly resistant to phage infection. Phages isolated from coinfected cultures were identified as recombinants of LP-048 and LP-125. Interestingly, recombination events occurred twice independently in a locus encoding two proteins putatively involved in DNA binding. We show that short-term coevolution of phages and their hosts can be utilized to obtain mutant and recombinant phages with adapted host ranges. These laboratory-evolved phages may be useful for limiting the emergence of phage resistance and for targeting strains that show general resistance to wild-type (WT) phages. IMPORTANCE Listeria monocytogenes is a life-threatening bacterial foodborne pathogen that can persist in food processing facilities for years. Phages can be used to control L. monocytogenes in food production, but phage-resistant bacterial subpopulations can regrow in phage-treated environments. Coevolution experiments were conducted on a Listeria phage-host system to provide insight into the genetic variation that emerges in both the phage and bacterial host under reciprocal selective pressure. As expected, mutations were identified in both phage and host, but additionally, recombination events were shown to have repeatedly occurred between closely related phages that coinfected L. monocytogenes. This study demonstrates that in vitro evolution of phages can be utilized to expand the host range and improve the long-term efficacy of phage-based control of L. monocytogenes. This approach may also be applied to other phage-host systems for applications in biocontrol, detection, and phage therapy.

scholarly journals In VitroandIn VivoActivities of Zinc Linolenate, a Selective Antibacterial Agent againstHelicobacter pylori

2019 ◽  
Vol 63 (6) ◽  
Author(s):  
Yanqiang Huang ◽  
Xudong Hang ◽  
Xueqing Jiang ◽  
Liping Zeng ◽  
Jia Jia ◽  
...  
Keyword(s):  
Gut Microbiota ◽  
Therapeutic Potential ◽  
Combined Therapy ◽  
Peptic Ulcers ◽  
Current Standard ◽  
Gastric Diseases ◽  
Content Type ◽  
H Pylori

ABSTRACTHelicobacter pyloriis a major global pathogen, and its infection represents a key factor in the etiology of various gastric diseases, including gastritis, peptic ulcers, and gastric carcinoma. The efficacy of current standard treatment forH. pyloriinfection including two broad-spectrum antibiotics is compromised by toxicity toward the gut microbiota and the development of drug resistance, which will likely only be resolved through novel and selective antibacterial strategies. Here, we synthesized a small molecule, zinc linolenate (ZnLla), and investigated its therapeutic potential for the treatment ofH. pyloriinfection. ZnLla showed effective antibacterial activity against standard strains and drug-resistant clinical isolates ofH. pyloriin vitrowith no development of resistance during continuous serial passaging. The mechanisms of ZnLla action againstH. pyloriinvolved the disruption of bacterial cell membranes and generation of reactive oxygen species. In mouse models of multidrug-resistantH. pyloriinfection, ZnLla showedin vivokilling efficacy comparable and superior to the triple therapy approach when use as a monotherapy and a combined therapy with omeprazole, respectively. Moreover, ZnLla treatment induces negligible toxicity against normal tissues and causes minimal effects on both the diversity and composition of the murine gut microbiota. Thus, the high degree of selectivity of ZnLla forH. pyloriprovides an attractive candidate for novel targeted anti-H. pyloritreatment.

scholarly journals cAMP and Vfr Control Exolysin Expression and Cytotoxicity of Pseudomonas aeruginosa Taxonomic Outliers

2018 ◽  
Vol 200 (12) ◽  
Author(s):  
Alice Berry ◽  
Kook Han ◽  
Julian Trouillon ◽  
Mylène Robert-Genthon ◽  
Michel Ragno ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Epithelial Cells ◽  
Transcriptional Activation ◽  
Opportunistic Pathogen ◽  
Secretion System ◽  
Intracellular Camp ◽  
Virulence Determinants ◽  
Cell Infection ◽  
Content Type

ABSTRACT The two-partner secretion system ExlBA, expressed by strains of Pseudomonas aeruginosa belonging to the PA7 group, induces hemorrhage in lungs due to disruption of host cellular membranes. Here we demonstrate that the exlBA genes are controlled by a pathway consisting of cAMP and the virulence factor regulator (Vfr). Upon interaction with cAMP, Vfr binds directly to the exlBA promoter with high affinity (equilibrium binding constant [ K eq ] of ≈2.5 nM). The exlB and exlA expression was diminished in the Vfr-negative mutant and upregulated with increased intracellular cAMP levels. The Vfr binding sequence in the exlBA promoter was mutated in situ , resulting in reduced cytotoxicity of the mutant, showing that Vfr is required for the exlBA expression during intoxication of epithelial cells. Vfr also regulates function of type 4 pili previously shown to facilitate ExlA activity on epithelial cells, which indicates that the cAMP/Vfr pathway coordinates these two factors needed for full cytotoxicity. As in most P. aeruginosa strains, the adenylate cyclase CyaB is the main provider of cAMP for Vfr regulation during both in vitro growth and eukaryotic cell infection. We discovered that the absence of functional Vfr in the reference strain PA7 is caused by a frameshift in the gene and accounts for its reduced cytotoxicity, revealing the conservation of ExlBA control by the CyaB-cAMP/Vfr pathway in P. aeruginosa taxonomic outliers. IMPORTANCE The human opportunistic pathogen Pseudomonas aeruginosa provokes severe acute and chronic human infections associated with defined sets of virulence factors. The main virulence determinant of P. aeruginosa taxonomic outliers is exolysin, a membrane-disrupting pore-forming toxin belonging to the two-partner secretion system ExlBA. In this work, we demonstrate that the conserved CyaB-cAMP/Vfr pathway controls cytotoxicity of outlier clinical strains through direct transcriptional activation of the exlBA operon. Therefore, despite the fact that the type III secretion system and exolysin are mutually exclusive in classical and outlier strains, respectively, these two major virulence determinants share similarities in their mechanisms of regulation.

scholarly journals The 5′ NAD Cap of RNAIII Modulates Toxin Production in Staphylococcus aureus Isolates

2019 ◽  
Vol 202 (6) ◽  
Author(s):  
Hector Gabriel Morales-Filloy ◽  
Yaqing Zhang ◽  
Gabriele Nübel ◽  
Shilpa Elizabeth George ◽  
Natalya Korn ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Secondary Structure ◽  
Quorum Sensing ◽  
Opportunistic Pathogen ◽  
Toxin Production ◽  
Dual Function ◽  
Content Type ◽  
The Stability

ABSTRACT Nicotinamide adenosine dinucleotide (NAD) has been found to be covalently attached to the 5′ ends of specific RNAs in many different organisms, but the physiological consequences of this modification are largely unknown. Here, we report the occurrence of several NAD-RNAs in the opportunistic pathogen Staphylococcus aureus. Most prominently, RNAIII, a central quorum-sensing regulator of this bacterium’s physiology, was found to be 5′ NAD capped in a range from 10 to 35%. NAD incorporation efficiency into RNAIII was found to depend in vivo on the −1 position of the P3 promoter. An increase in RNAIII’s NAD content led to a decreased expression of alpha- and delta-toxins, resulting in reduced cytotoxicity of the modified strains. These effects seem to be caused neither by changes in RNAIII’s secondary structure nor by a different translatability upon NAD attachment, as indicated by unaltered patterns in in vitro chemical probing and toeprinting experiments. Even though we did not observe any effect of this modification on RNAIII’s secondary structure or translatability in vitro, additional unidentified factors might account for the modulation of exotoxins in vivo. Ultimately, the study constitutes a step forward in the discovery of new roles of the NAD molecule in bacteria. IMPORTANCE Numerous organisms, including bacteria, are endowed with a 5′ NAD cap in specific RNAs. While the presence of the 5′ NAD cap modulates the stability of the modified RNA species, a significant biological function and phenotype have not been assigned so far. Here, we show the presence of a 5′ NAD cap in RNAIII from S. aureus, a dual-function regulatory RNA involved in quorum-sensing processes and regulation of virulence factor expression. We also demonstrate that altering the natural NAD modification ratio of RNAIII leads to a decrease in exotoxin production, thereby modulating the bacterium’s virulence. Our work unveils a new layer of regulation of RNAIII and the agr system that might be linked to the redox state of the NAD molecule in the cell.

scholarly journals Hitchhiking with Nature: Snake Venom Peptides to Fight Cancer and Superbugs

Toxins ◽  
2020 ◽  
Vol 12 (4) ◽  
pp. 255 ◽  
Author(s):  
Clara Pérez-Peinado ◽  
Sira Defaus ◽  
David Andreu
Keyword(s):  
Therapeutic Potential ◽  
State Of The Art ◽  
Resistant Bacteria ◽  
Snake Venoms ◽  
Antibiotic Resistant ◽  
Anticancer Peptides ◽  
Therapeutic Development ◽  
Drug Leads

For decades, natural products in general and snake venoms (SV) in particular have been a rich source of bioactive compounds for drug discovery, and they remain a promising substrate for therapeutic development. Currently, a handful of SV-based drugs for diagnosis and treatment of various cardiovascular disorders and blood abnormalities are on the market. Likewise, far more SV compounds and their mimetics are under investigation today for diverse therapeutic applications, including antibiotic-resistant bacteria and cancer. In this review, we analyze the state of the art regarding SV-derived compounds with therapeutic potential, focusing on the development of antimicrobial and anticancer drugs. Specifically, information about SV peptides experimentally validated or predicted to act as antimicrobial and anticancer peptides (AMPs and ACPs, respectively) has been collected and analyzed. Their principal activities both in vitro and in vivo, structures, mechanisms of action, and attempts at sequence optimization are discussed in order to highlight their potential as drug leads.

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