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 Loss of function mutations inepaRconfer resistance to phage NPV1 infection inEnterococcus faecalisOG1RF

2018 ◽  
Author(s):  
Khang Ho ◽  
Wenwen Huo ◽  
Savannah Pas ◽  
Ryan Dao ◽  
Kelli L. Palmer
Keyword(s):  
High Frequency ◽  
Opportunistic Pathogen ◽  
Phage Resistance ◽  
Loss Of Function ◽  
Genetic Determinants ◽  
Human Gastrointestinal Tract ◽  
Antibiotic Resistant ◽  
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 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 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 Mutations in fbiD (Rv2983) as a Novel Determinant of Resistance to Pretomanid and Delamanid in Mycobacterium tuberculosis

2020 ◽  
Vol 65 (1) ◽  
pp. e01948-20
Author(s):  
Dalin Rifat ◽  
Si-Yang Li ◽  
Thomas Ioerger ◽  
Keshav Shah ◽  
Jean-Philippe Lanoix ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Clinical Evidence ◽  
Clinical Samples ◽  
Loss Of Function ◽  
Wild Type ◽  
Content Type ◽  
Solid Media ◽  
High Level ◽  
Level Resistance

ABSTRACTThe nitroimidazole prodrugs delamanid and pretomanid comprise one of only two new antimicrobial classes approved to treat tuberculosis (TB) in 50 years. Prior in vitro studies suggest a relatively low barrier to nitroimidazole resistance in Mycobacterium tuberculosis, but clinical evidence is limited to date. We selected pretomanid-resistant M. tuberculosis mutants in two mouse models of TB using a range of pretomanid doses. The frequency of spontaneous resistance was approximately 10−5 CFU. Whole-genome sequencing of 161 resistant isolates from 47 mice revealed 99 unique mutations, of which 91% occurred in 1 of 5 genes previously associated with nitroimidazole activation and resistance, namely, fbiC (56%), fbiA (15%), ddn (12%), fgd (4%), and fbiB (4%). Nearly all mutations were unique to a single mouse and not previously identified. The remaining 9% of resistant mutants harbored mutations in Rv2983 (fbiD), a gene not previously associated with nitroimidazole resistance but recently shown to be a guanylyltransferase necessary for cofactor F420 synthesis. Most mutants exhibited high-level resistance to pretomanid and delamanid, although Rv2983 and fbiB mutants exhibited high-level pretomanid resistance but relatively small changes in delamanid susceptibility. Complementing an Rv2983 mutant with wild-type Rv2983 restored susceptibility to pretomanid and delamanid. By quantifying intracellular F420 and its precursor Fo in overexpressing and loss-of-function mutants, we provide further evidence that Rv2983 is necessary for F420 biosynthesis. Finally, Rv2983 mutants and other F420H2-deficient mutants displayed hypersusceptibility to some antibiotics and to concentrations of malachite green found in solid media used to isolate and propagate mycobacteria from clinical samples.

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 The Redox Cofactor F420Protects Mycobacteria from Diverse Antimicrobial Compounds and Mediates a Reductive Detoxification System

2016 ◽  
Vol 82 (23) ◽  
pp. 6810-6818 ◽  
Author(s):  
Thanavit Jirapanjawat ◽  
Blair Ney ◽  
Matthew C. Taylor ◽  
Andrew C. Warden ◽  
Shahana Afroze ◽  
...  
Keyword(s):  
Antimicrobial Resistance ◽  
Mycobacterium Smegmatis ◽  
Antimicrobial Compounds ◽  
Loss Of Function ◽  
Detoxifying Enzymes ◽  
Content Type ◽  
Wide Range ◽  
Detoxification System ◽  
Mutant Strains

ABSTRACTA defining feature of mycobacterial redox metabolism is the use of an unusual deazaflavin cofactor, F420. This cofactor enhances the persistence of environmental and pathogenic mycobacteria, including after antimicrobial treatment, although the molecular basis for this remains to be understood. In this work, we explored our hypothesis that F420enhances persistence by serving as a cofactor in antimicrobial-detoxifying enzymes. To test this, we performed a series of phenotypic, biochemical, and analytical chemistry studies in relation to the model soil bacteriumMycobacterium smegmatis. Mutant strains unable to synthesize or reduce F420were found to be more susceptible to a wide range of antibiotic and xenobiotic compounds. Compounds from three classes of antimicrobial compounds traditionally resisted by mycobacteria inhibited the growth of F420mutant strains at subnanomolar concentrations, namely, furanocoumarins (e.g., methoxsalen), arylmethanes (e.g., malachite green), and quinone analogues (e.g., menadione). We demonstrated that promiscuous F420H2-dependent reductases directly reduce these compounds by a mechanism consistent with hydride transfer. Moreover,M. smegmatisstrains unable to make F420H2lost the capacity to reduce and detoxify representatives of the furanocoumarin and arylmethane compound classes in whole-cell assays. In contrast, mutant strains were only slightly more susceptible to clinical antimycobacterials, and this appeared to be due to indirect effects of F420loss of function (e.g., redox imbalance) rather than loss of a detoxification system. Together, these data show that F420enhances antimicrobial resistance in mycobacteria and suggest that one function of the F420H2-dependent reductases is to broaden the range of natural products that mycobacteria and possibly other environmental actinobacteria can reductively detoxify.IMPORTANCEThis study reveals that a unique microbial cofactor, F420, is critical for antimicrobial resistance in the environmental actinobacteriumMycobacterium smegmatis. We show that a superfamily of redox enzymes, the F420H2-dependent reductases, can reduce diverse antimicrobialsin vitroandin vivo.M. smegmatisstrains unable to make or reduce F420become sensitive to inhibition by these antimicrobial compounds. This suggests that mycobacteria have harnessed the unique properties of F420to reduce structurally diverse antimicrobials as part of the antibiotic arms race. The F420H2-dependent reductases that facilitate this process represent a new class of antimicrobial-detoxifying enzymes with potential applications in bioremediation and biocatalysis.

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 Drug Susceptibility Profiling and Genetic Determinants of Drug Resistance inMycobacterium kansasii

2018 ◽  
Vol 62 (4) ◽  
pp. e01788-17 ◽  
Author(s):  
Zofia Bakuła ◽  
Magdalena Modrzejewska ◽  
Lian Pennings ◽  
Małgorzata Proboszcz ◽  
Aleksandra Safianowska ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Target Genes ◽  
Regulatory Region ◽  
Drug Susceptibility ◽  
Dilution Method ◽  
Broth Microdilution ◽  
Genetic Determinants ◽  
Content Type ◽  
Microdilution Method

ABSTRACTVery few studies have examined drug susceptibility ofMycobacterium kansasii, and they involve a limited number of strains. The purpose of this study was to determine drug susceptibility profiles ofM. kansasiiisolates representing a spectrum of species genotypes (subtypes) with two different methodologies, i.e., broth microdilution and Etest assays. To confirm drug resistance, drug target genes were sequenced. A collection of 85M. kansasiiisolates, including representatives of eight different subtypes (I to VI, I/II, and IIB) from eight countries, was used. Drug susceptibility against 13 and 8 antimycobacterial agents was tested by using broth microdilution and Etest, respectively. For drug-resistant or high-MIC isolates, eight structural genes (rrl,katG,inhA,embB,rrs,rpsL,gyrA, andgyrB) and one regulatory region (embCA) were PCR amplified and sequenced in the search for resistance-associated mutations. All isolates tested were susceptible to rifampin (RIF), amikacin (AMK), co-trimoxazole (SXT), rifabutin (RFB), moxifloxacin (MXF), and linezolid (LZD) according to the microdilution method. Resistance to ethambutol (EMB), ciprofloxacin (CIP), and clarithromycin (CLR) was found in 83 (97.7%), 17 (20%), and 1 (1.2%) isolate, respectively. The calculated concordance between the Etest and dilution method was 22.6% for AMK, 4.8% for streptomycin (STR), 3.2% for CLR, and 1.6% for RIF. For EMB, INH, and SXT, not even a single MIC value determined by one method equaled that by the second method. The only mutations disclosed were A2266C transversion at therrlgene (CLR-resistant strain) and A128G transition at therpsLgene (strain with STR MIC of >64 mg/liter). In conclusion, eight drugs, including RIF, CLR, AMK, SXT, RFB, MXF, LZD, and ethionamide (ETO), showed highin vitroactivity againstM. kansasiiisolates. Discrepancies of the results between the reference microdilution method and Etest preclude the use of the latter for drug susceptibility determination inM. kansasii. Drug resistance inM. kansasiimay have different genetic determinants than resistance to the same drugs inM. tuberculosis.

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 Putative Inv Is Essential for Basolateral Invasion of Caco-2 Cells and Acts Synergistically with OmpA To AffectIn VitroandIn VivoVirulence of Cronobacter sakazakii ATCC 29544

2014 ◽  
Vol 82 (5) ◽  
pp. 1755-1765 ◽  
Author(s):  
Dilini Chandrapala ◽  
Kyumson Kim ◽  
Younho Choi ◽  
Amal Senevirathne ◽  
Dong-Hyun Kang ◽  
...  
Keyword(s):  
Outer Membrane Proteins ◽  
Opportunistic Pathogen ◽  
Additive Effect ◽  
Neonatal Meningitis ◽  
Cronobacter Sakazakii ◽  
Content Type ◽  
Enteric Infections ◽  
Culture Models

ABSTRACTCronobacter sakazakiiis an opportunistic pathogen that causes neonatal meningitis and necrotizing enterocolitis. Its interaction with intestinal epithelium is important in the pathogenesis of enteric infections. In this study, we investigated the involvement of theinvgene in the virulence ofC. sakazakiiATCC 29544in vitroandin vivo. Sequence analysis ofC. sakazakiiATCC 29544invrevealed that it is different from otherC. sakazakiiisolates. In various cell culture models, an Δinvdeletion mutant showed significantly lowered invasion efficiency, which was restored upon genetic complementation. Studying invasion potentials using tight-junction-disrupted Caco-2 cells suggested that theinvgene product mediates basolateral invasion ofC. sakazakiiATCC 29544. In addition, comparison of invasion potentials of double mutant (ΔompA Δinv) and single mutants (ΔompAand Δinv) provided evidence for an additive effect of the two putative outer membrane proteins. Finally, the importance ofinvand the additive effect of putative Inv and OmpA were also proven in anin vivorat pup model. This report is the first to demonstrate two proteins working synergisticallyin vitro, as well asin vivoinC. sakazakiipathogenesis.

scholarly journals Enterococcus faecalis 6-Phosphogluconolactonase Is Required for Both Commensal and Pathogenic Interactions with Manduca sexta

2014 ◽  
Vol 83 (1) ◽  
pp. 396-404 ◽  
Author(s):  
Jonathan F. Holt ◽  
Megan R. Kiedrowski ◽  
Kristi L. Frank ◽  
Jing Du ◽  
Changhui Guan ◽  
...  
Keyword(s):  
Enterococcus Faecalis ◽  
Manduca Sexta ◽  
Wild Type ◽  
Genetic Determinants ◽  
Content Type ◽  
Polystyrene Plate ◽  
Insect Gut ◽  
Insight Into

Enterococcus faecalisis a commensal and pathogen of humans and insects. InManduca sexta,E. faecalisis an infrequent member of the commensal gut community, but its translocation to the hemocoel results in a commensal-to-pathogen switch. To investigateE. faecalisfactors required for commensalism, we identifiedE. faecalisgenes that are upregulated in the gut ofM. sextausing recombinase-basedin vivoexpression technology (RIVET). The RIVET screen produced 113 clones, from which we identified 50 genes that are more highly expressed in the insect gut than in culture. The most frequently recovered gene was locus OG1RF_11582, which encodes a 6-phosphogluconolactonase that we designatedpglA. ApglAdeletion mutant was impaired in both pathogenesis and gut persistence inM. sextaand produced enhanced biofilms compared with the wild type in anin vitropolystyrene plate assay. Mutation of four other genes identified by RIVET did not affect persistence in caterpillar guts but led to impaired pathogenesis. This is the first identification of genetic determinants forE. faecaliscommensal and pathogenic interactions withM. sexta. Bacterial factors identified in this model system may provide insight into colonization or persistence in other host-associated microbial communities and represent potential targets for interventions to preventE. faecalisinfections.

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