scholarly journals Death Becomes Them: Bacterial Community Dynamics and Stilbene Antibiotic Production in Cadavers of Galleria mellonella Killed by Heterorhabditis and Photorhabdus spp.

2016 ◽  
Vol 82 (19) ◽  
pp. 5824-5837 ◽  
Author(s):  
Amanda C. Wollenberg ◽  
Tanush Jagdish ◽  
Greg Slough ◽  
Megan E. Hoinville ◽  
Michael S. Wollenberg
Keyword(s):  
Entomopathogenic Nematodes ◽  
Community Dynamics ◽  
Resource Competition ◽  
Galleria Mellonella ◽  
Insect Larvae ◽  
Content Type ◽  
Bacterial Community Dynamics ◽  
Insect Cadaver ◽  
Generation Sequencing

ABSTRACTInsect larvae killed by entomopathogenic nematodes are thought to contain bacterial communities dominated by a single bacterial genus, that of the nematode's bacterial symbiont. In this study, we used next-generation sequencing to profile bacterial community dynamics in greater wax moth (Galleria mellonella) larvae cadavers killed byHeterorhabditisnematodes and theirPhotorhabdussymbionts. We found that, althoughPhotorhabdusstrains did initially displace anEnterococcus-dominated community present in uninfectedG. mellonellainsect larvae, the cadaver community was not static. Twelve days postinfection,Photorhabdusshared the cadaver withStenotrophomonasspecies. Consistent with this result,Stenotrophomonasstrains isolated from infected cadavers were resistant toPhotorhabdus-mediated toxicity in solid coculture assays. We isolated and characterized aPhotorhabdus-produced antibiotic fromG. mellonellacadavers, produced it synthetically, and demonstrated that both the natural and synthetic compounds decreasedG. mellonella-associatedEnterococcusgrowth, but notStenotrophomonasgrowth,in vitro. Finally, we showed that theStenotrophomonasstrains described here negatively affectedPhotorhabdusgrowthin vitro. Our results add an important dimension to a broader understanding ofHeterorhabditis-Photorhabdusbiology and also demonstrate that interspecific bacterial competition likely characterizes even a theoretically monoxenic environment, such as aHeterorhabditis-Photorhabdus-parasitized insect cadaver.IMPORTANCEUnderstanding, and eventually manipulating, both human and environmental health depends on a complete accounting of the forces that act on and shape microbial communities. One of these underlying forces is hypothesized to be resource competition. A resource that has received little attention in the general microbiological literature, but likely has ecological and evolutionary importance, is dead/decaying multicellular organisms. Metazoan cadavers, including those of insects, are ephemeral and nutrient-rich environments, where resource competition might shape interspecific macrobiotic and microbiotic interactions. This study is the first to use a next-generation sequencing approach to study the community dynamics of bacteria within a model insect cadaver system: insect larvae parasitized by entomopathogenic nematodes and their bacterial symbionts. By integrating bioinformatic, biochemical, and classicin vitromicrobiological approaches, we have provided mechanistic insight into how antibiotic-mediated bacterial interactions may shape community dynamics within insect cadavers.

scholarly journals Biological Impact of a Large-Scale Genomic Inversion That Grossly Disrupts the Relative Positions of the Origin and Terminus Loci of theStreptococcus pyogenesChromosome

2019 ◽  
Vol 201 (17) ◽  
Author(s):  
Dragutin J. Savic ◽  
Scott V. Nguyen ◽  
Kimberly McCullor ◽  
W. Michael McShan
Keyword(s):  
Dna Sequences ◽  
Parental Strain ◽  
Large Scale ◽  
Galleria Mellonella ◽  
Acute Infection ◽  
Relative Length ◽  
Published Data ◽  
Rich Medium ◽  
Content Type

ABSTRACTA large-scale genomic inversion encompassing 0.79 Mb of the 1.816-Mb-longStreptococcus pyogenesserotype M49 strain NZ131 chromosome spontaneously occurs in a minor subpopulation of cells, and in this report genetic selection was used to obtain a stable lineage with this chromosomal rearrangement. This inversion, which drastically displaces theorisite relative to the terminus, changes the relative length of the replication arms so that one replichore is approximately 0.41 Mb while the other is about 1.40 Mb in length. Genomic reversion to the original chromosome constellation is not observed in PCR-monitored analyses after 180 generations of growth in rich medium. Compared to the parental strain, the inversion surprisingly demonstrates a nearly identical growth pattern in the first phase of the exponential phase, but differences do occur when resources in the medium become limited. When cultured separately in rich medium during prolonged stationary phase or in an experimental acute infection animal model (Galleria mellonella), the parental strain and the invertant have equivalent survival rates. However, when they are coincubated together, bothin vitroandin vivo, the survival of the invertant declines relative to the level for the parental strain. The accompanying aspect of the study suggests that inversions taking place nearoriCalways happen to secure the linkage oforiCto DNA sequences responsible for chromosome partition. The biological relevance of large-scale inversions is also discussed.IMPORTANCEBased on our previous work, we created to our knowledge the largest asymmetric inversion, covering 43.5% of theS. pyogenesgenome. In spite of a drastic replacement of origin of replication and the unbalanced size of replichores (1.4 Mb versus 0.41 Mb), the invertant, when not challenged with its progenitor, showed impressive vitality for growthin vitroand in pathogenesis assays. The mutant supports the existing idea that slightly deleterious mutations can provide the setting for secondary adaptive changes. Furthermore, comparative analysis of the mutant with previously published data strongly indicates that even large genomic rearrangements survive provided that the integrity of theoriCand the chromosome partition cluster is preserved.

scholarly journals Dissecting the Structure-Function Relationship of a Fungicidal Peptide Derived from the Constant Region of Human Immunoglobulins

2016 ◽  
Vol 60 (4) ◽  
pp. 2435-2442 ◽  
Author(s):  
Tecla Ciociola ◽  
Thelma A. Pertinhez ◽  
Laura Giovati ◽  
Martina Sperindè ◽  
Walter Magliani ◽  
...  
Keyword(s):  
Self Assembly ◽  
Galleria Mellonella ◽  
Critical Role ◽  
Yeast Cells ◽  
Therapeutic Effects ◽  
Constant Region ◽  
Content Type ◽  
Complementarity Determining Regions

ABSTRACTSynthetic peptides encompassing sequences related to the complementarity-determining regions of antibodies or derived from their constant region (Fc peptides) were proven to exert differential antimicrobial, antiviral, antitumor, and/or immunomodulatory activitiesin vitroand/orin vivo, regardless of the specificity and isotype of the parental antibody. Alanine substitution derivatives of these peptides exhibited unaltered, increased, or decreased candidacidal activitiesin vitro. The bioactive IgG-derived Fc N10K peptide (NQVSLTCLVK) spontaneously self-assembles, a feature previously recognized as relevant for the therapeutic activity of another antibody-derived peptide. We evaluated the contribution of each residue to the peptide self-assembling capability by circular-dichroism spectroscopy. The interaction of the N10K peptide and its derivatives withCandida albicanscells was studied by confocal, transmission, and scanning electron microscopy. The apoptosis and autophagy induction profiles in yeast cells treated with the peptides were evaluated by flow cytometry, and the therapeutic efficacy against candidal infection was studied in aGalleria mellonellamodel. Overall, the results indicate a critical role for some residues in the self-assembly process and a correlation of that capability with the candidacidal activities of the peptidesin vitroand their therapeutic effectsin vivo.

scholarly journals Activity of Telavancin against Staphylococcus aureus Isolates, Including Those with Decreased Susceptibility to Ceftaroline, from Cystic Fibrosis Patients

2018 ◽  
Vol 62 (9) ◽  
Author(s):  
Melanie Roch ◽  
Maria Celeste Varela ◽  
Agustina Taglialegna ◽  
Warren E. Rose ◽  
Adriana E. Rosato
Keyword(s):  
Cystic Fibrosis ◽  
Staphylococcus Aureus ◽  
Galleria Mellonella ◽  
Therapeutic Option ◽  
The United States ◽  
Infection Model ◽  
Content Type ◽  
Complicated Skin ◽  
Hospital Acquired

ABSTRACT Methicillin-resistant Staphylococcus aureus (MRSA) acquisition in cystic fibrosis (CF) patients confers a clinical outcome worse than that in non-CF patients with an increased rate of declined lung function. Telavancin, an approved lipoglycopeptide used to treat infections due to S. aureus, has a dual mode of action causing inhibition of peptidoglycan synthesis and membrane depolarization. MRSA infections in CF patients remain an important problem with no foreseeable decline in prevalence rates. Although telavancin is currently in clinical use for the treatment of complicated skin infections and hospital-acquired pneumonia, the activity against S. aureus infections in CF patients has not been investigated. In this work, we studied the activity of telavancin against CF patient-derived S. aureus strains collected from geographically diverse CF centers in the United States. We found that the telavancin MIC90 was 0.06 μg/ml, 8-fold lower than the ceftaroline or daptomycin MIC90 and 25-fold lower than the linezolid and vancomycin MIC90. We demonstrate that telavancin at serum free concentrations has rapid bactericidal activity, with a decrease of more than 3 log10 CFU/ml being achieved during the first 4 to 6 h of treatment, performing better in this assay than vancomycin and ceftaroline, including against S. aureus strains resistant to ceftaroline. Telavancin resistance was infrequent (0.3%), although we found that it can occur in vitro in both CF- and non-CF patient-derived S. aureus strains by progressive passages with subinhibitory concentrations. Genetic analysis of telavancin-resistant in vitro mutants showed gene polymorphisms in cell wall and virulence genes and increased survival in a Galleria mellonella infection model. Thus, we conclude that telavancin represents a promising therapeutic option for infections in CF patients with potent in vitro activity and a low resistance development potential.

scholarly journals Implications of the EUCAST Trailing Phenomenon inCandida tropicalisfor theIn VivoSusceptibility in Invertebrate and Murine Models

2018 ◽  
Vol 62 (12) ◽  
Author(s):  
K. M. T. Astvad ◽  
D. Sanglard ◽  
E. Delarze ◽  
R. K. Hare ◽  
M. C. Arendrup
Keyword(s):  
Susceptibility Testing ◽  
Galleria Mellonella ◽  
Growth Control ◽  
Resistant Isolate ◽  
Murine Models ◽  
Wild Type ◽  
Content Type ◽  
Gradual Loss

ABSTRACTCandida tropicalisisolates often display reduced but persistent growth (trailing) over a broad fluconazole concentration range during EUCAST susceptibility testing. Whereas weak trailing (<25% of the positive growth control) is common and found not to impair fluconazole efficacy, we investigated if more pronounced trailing impacted treatment efficacy. Fluconazole efficacy against two weakly (≤25% growth), two moderately (26% to 50% growth), and one heavily (>70% growth) trailing resistant isolate and one resistant (100% growth) isolate were investigatedin vitroandin vivo(in aGalleria mellonellasurvival model and two nonlethal murine models).CDR1expression levels andERG11sequences were characterized. The survival in fluconazole-treatedG. mellonellawas inversely correlated with the degree of trailing (71% to 9% survival in treatment groups). In mice, resistant and heavily trailing isolates responded poorly to fluconazole treatment.CDR1expression was significantly higher in trailing and resistant isolates than in wild-type isolates (1.4-fold to 10-fold higher). All isolates exhibitedERG11wild-type alleles. Heavily trailing isolates were less responsive to fluconazole in allin vivomodels, indicating an impact on fluconazole efficacy.CDR1upregulation may have contributed to the observed differences. Moderately trailing isolates responded less well to fluconazole in larvae only. This confirms clinical data suggesting fluconazole is effective against infections with such isolates in less severely ill patients and supports the current 50% growth endpoint for susceptibility testing. However, it is still unclear if the gradual loss of efficacy observed for moderately trailing isolates in the larva model may be a reason for concern in selected vulnerable patient populations.

Influence of temperature on the infectivity of entomopathogenic nematodes (Steinernema and Heterorhabditis spp.) to larvae and pupae of the vine weevil Otiorhynchus sulcatus (Coleoptera: Curculionidae)

Nematology ◽  
2000 ◽  
Vol 2 (3) ◽  
pp. 309-317 ◽  
Author(s):  
Steve Long ◽  
John Fenlon ◽  
Paul Richardson
Keyword(s):  
Competing Risks ◽  
Entomopathogenic Nematodes ◽  
Galleria Mellonella ◽  
Commercial Product ◽  
Effect Of Temperature ◽  
Heterorhabditis Megidis ◽  
Otiorhynchus Sulcatus ◽  
The Uk

AbstractThe susceptibilities of early and late instar vine weevil larvae and pupae to three species of entomopathogenic nematodes, indigenous to the UK, were tested in a series of bioassays. Steinernema kraussei (isolates L017 and L137), S. feltiae (the commercial product Nemasys®) and Heterorhabditis megidis (the commercial product Nemasys® H, reared both in vivo in Galleria mellonella larvae and in vitro), were tested at 6, 10 and 18°C for 2 weeks (early instars of O. sulcatus) or 3 weeks (late instars and pupae of O. sulcatus). Nematodes were applied to over 3800 larvae or pupae and there were over 400 untreated controls. Each insect was examined subsequently to determine mortality, and parasitised specimens were dissected to establish whether adult nematodes had developed. Differences in pathogenicity between H. megidis reared in vitro and in vivo were demonstrated. S. kraussei (L137) was consistently the most virulent nematode isolate at low temperatures. The results revealed a significant (P < 0.001) effect of temperature on small larvae of O. sulcatus, but also showed differential levels of mortality, not due to nematodes, for both small larvae and pupae. The use of Abbott's correction for control mortality is challenged and the validity of competing risks theory examined.In einer Reihe von Biotests wurde die Anfälligkeit von frühen und späten Larvenstadien sowie Puppen des Rüsselkäfers Otiorhynchus sulcatus gegenüber drei in UK einheimischen entomopathogenen Nematoden untersucht. Steinernema kraussei (isolate L017 und L137), S. feltiae (Handelsprodukt Nemasys®) und Heterorhabditis megidis (Handelsprodukt Nemasys® H, beide in vivo an Larven von Galleria mellonella und in vitro gezüchtet) wurden bei 6, 10 and 18°C für zwei Wochen (frühe Stadien von O. sulcatus) oder drei Wochen (späte Stadien und Puppen von O. sulcatus) geprüft. Über 3800 Larven oder Puppen wurden mit Nematoden behandelt, daneben gab es über 400 unbehandelte Kontrollen. Anschliessend wurde jedes Insekt untersucht, um die Mortalität zu bestimmen. Parasitierte Exemplare wurden aufpräpariert um festzustellen, ob sich adulte Nematoden entwickelt hatten. Zwischen in vitro und in vivo kultivierten H. megidis konnten Unterschiede in der Pathogenität festgestellt werden. S. kraussei (L137) war bei niedrigen Temperaturen durchgehend das virulenteste Isolat. Die Ergebnisse zeigten eine signifikante (P < 0.001) Wirkung der Temperatur auf kleine Larven von O. sulcatus. Sie zeigten für kleine Larven und Puppen aber auch unterschiedliche Mortlitätsgrade an, die nicht auf Nematoden zurückgingen. Die Anwendung von Abbott's Korrektur zur Prüfung der Mortalität wird kritisch hinterfragt, die Gültigkeit der Theorie der “competing risks” wird geprüft.

scholarly journals Synergistic Efficacy of Aedes aegypti Antimicrobial Peptide Cecropin A2 and Tetracycline against Pseudomonas aeruginosa

2017 ◽  
Vol 61 (7) ◽  
Author(s):  
Zhaojun Zheng ◽  
Nagendran Tharmalingam ◽  
Qingzhong Liu ◽  
Elamparithi Jayamani ◽  
Wooseong Kim ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Aedes Aegypti ◽  
Mammalian Cells ◽  
Galleria Mellonella ◽  
Multidrug Resistant ◽  
Resistant Bacteria ◽  
Synergistic Activity ◽  
Content Type

ABSTRACT The increasing prevalence of antibiotic resistance has created an urgent need for alternative drugs with new mechanisms of action. Antimicrobial peptides (AMPs) are promising candidates that could address the spread of multidrug-resistant bacteria, either alone or in combination with conventional antibiotics. We studied the antimicrobial efficacy and bactericidal mechanism of cecropin A2, a 36-residue α-helical cationic peptide derived from Aedes aegypti cecropin A, focusing on the common pathogen Pseudomonas aeruginosa. The peptide showed little hemolytic activity and toxicity toward mammalian cells, and the MICs against most clinical P. aeruginosa isolates were 32 to 64 μg/ml, and its MICs versus other Gram-negative bacteria were 2 to 32 μg/ml. Importantly, cecropin A2 demonstrated synergistic activity against P. aeruginosa when combined with tetracycline, reducing the MICs of both agents by 8-fold. The combination was also effective in vivo in the P. aeruginosa/Galleria mellonella model (P < 0.001). We found that cecropin A2 bound to P. aeruginosa lipopolysaccharides, permeabilized the membrane, and interacted with the bacterial genomic DNA, thus facilitating the translocation of tetracycline into the cytoplasm. In summary, the combination of cecropin A2 and tetracycline demonstrated synergistic antibacterial activity against P. aeruginosa in vitro and in vivo, offering an alternative approach for the treatment of P. aeruginosa infections.

scholarly journals Two Novel Bacteriophages Improve Survival in Galleria mellonella Infection and Mouse Acute Pneumonia Models Infected with Extensively Drug-Resistant Pseudomonas aeruginosa

2019 ◽  
Vol 85 (9) ◽  
Author(s):  
Jongsoo Jeon ◽  
Dongeun Yong
Keyword(s):  
Pseudomonas Aeruginosa ◽  
In Silico ◽  
Galleria Mellonella ◽  
Drug Resistant ◽  
Content Type ◽  
Bacteriolytic Activity ◽  
Extensively Drug Resistant ◽  
Acute Pneumonia

ABSTRACT Extensively drug-resistant Pseudomonas aeruginosa (XDR-PA) is a life-threatening pathogen that causes serious global problems. Here, we investigated two novel P. aeruginosa bacteriophages (phages), Bϕ-R656 and Bϕ-R1836, in vitro, in silico, and in vivo to evaluate the potential of phage therapy to control XDR-PA clinical strains. Bϕ-R656 and Bϕ-R1836 belong to the Siphoviridae family and exhibited broad host ranges which could lyse 18 (64%) and 14 (50%) of the 28 XDR-PA strains. In addition, the two phages showed strong bacteriolytic activity against XDR-PA host strains from pneumonia patients. The whole genomes of Bϕ-R656 and Bϕ-R1836 have linear double-stranded DNA of 60,919 and 37,714 bp, respectively. The complete sequence of Bϕ-R656 had very low similarity to the previously discovered P. aeruginosa phages in GenBank, but phage Bϕ-R1836 exhibited 98% and 91% nucleotide similarity to Pseudomonas phages YMC12/01/R24 and PA1/KOR/2010, respectively. In the two in vivo infection models, treatment with Bϕ-R656 and Bϕ-R1836 enhanced the survival of Galleria mellonella larvae (50% and 60%, respectively) at 72 h postinfection and pneumonia-model mice (66% and 83%, respectively) at 12 days postinfection compared with untreated controls. Treatment with Bϕ-R656 or Bϕ-R1836 also significantly decreased the bacterial load in the lungs of the mouse pneumonia model (>6 log10 CFU and >4 log10 CFU, respectively) on day 5. IMPORTANCE In this study, two novel P. aeruginosa phages, Bϕ-R656 and Bϕ-R1836, were evaluated in vitro, in silico, and in vivo for therapeutic efficacy and safety as an alternative antibacterial agent to control XDR-PA strains collected from pneumonia patients. Both phages exhibited potent bacteriolytic activity and greatly improved survival in G. mellonella larva infection and a mouse acute pneumonia model. Based on these results, we strongly predict that these two new phages could be used as fast-acting and safe alternative biological weapons against XDR-PA infections.

scholarly journals Extracellular Vesicles from Aspergillus flavus Induce M1 Polarization In Vitro

mSphere ◽  
2020 ◽  
Vol 5 (3) ◽  
Author(s):  
Verônica S. Brauer ◽  
André M. Pessoni ◽  
Tamires A. Bitencourt ◽  
Renato G. de Paula ◽  
Liliana de Oliveira Rocha ◽  
...  
Keyword(s):  
Fungal Infection ◽  
Extracellular Vesicles ◽  
Aspergillus Flavus ◽  
Crucial Role ◽  
Galleria Mellonella ◽  
Biologically Active ◽  
Common Cause ◽  
Content Type ◽  
M1 Macrophage

ABSTRACT Aspergillus flavus, a ubiquitous and saprophytic fungus, is the second most common cause of aspergillosis worldwide. Several mechanisms contribute to the establishment of the fungal infection. Extracellular vesicles (EVs) have been described as “virulence factor delivery bags” in several fungal species, demonstrating a crucial role during the infection. In this study, we evaluated production of A. flavus EVs and their immunomodulatory functions. We verified that A. flavus EVs induce macrophages to produce inflammatory mediators, such as nitric oxide, tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6), and IL-1β. Furthermore, the A. flavus EVs enhance phagocytosis and killing by macrophages and induce M1 macrophage polarization in vitro. In addition, a prior inoculation of A. flavus EVs in Galleria mellonella larvae resulted in a protective effect against the fungal infection. Our findings suggest that A. flavus EVs are biologically active and affect the interaction between A. flavus and host immune cells, priming the innate immune system to eliminate the fungal infection. Collectively, our results suggest that A. flavus EVs play a crucial role in aspergillosis. IMPORTANCE Immunocompromised patients are susceptible to several fungal infections. The genus Aspergillus can cause increased morbidity and mortality. Developing new therapies is essential to understand the fungal biology mechanisms. Fungal EVs carry important virulence factors, thus playing pivotal roles in fungal pathophysiology. No study to date has reported EV production by Aspergillus flavus, a fungus considered to be the second most common cause of aspergillosis and relevant food contaminator found worldwide. In this study, we produced A. flavus EVs and evaluated the in vitro immunomodulatory effects of EVs on bone marrow-derived macrophages (BMDMs) and in vivo effects in a Galleria mellonella model.

scholarly journals Promoter Variation and Gene Expression of mcr-1 -Harboring Plasmids in Clinical Isolates of Escherichia coli and Klebsiella pneumoniae from a Chinese Hospital

2018 ◽  
Vol 62 (5) ◽  
Author(s):  
Bin Huang ◽  
Yuting He ◽  
Xingyan Ma ◽  
Renxin Cai ◽  
Jianming Zeng ◽  
...  
Keyword(s):  
Gene Expression ◽  
Escherichia Coli ◽  
Klebsiella Pneumoniae ◽  
Tertiary Care ◽  
Care Hospital ◽  
Content Type ◽  
Sequence Variations ◽  
Generation Sequencing ◽  
Gene Expression Levels

ABSTRACT Next-generation sequencing of 6 mcr-1 -harboring Escherichia coli and Klebsiella pneumoniae isolates collected from a tertiary care hospital in China revealed significant sequence variations in the regions flanking the mcr-1 gene. While sequence variations significantly affected the expression and promoter activity of mcr-1 , the mcr-1 gene expression levels did not correlate with the in vitro colistin resistance levels, which warrants further in-depth investigations.

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