Prospecting Peptides Isolated From Black Soldier Fly (Diptera: Stratiomyidae) With Antimicrobial Activity Against Helicobacter pylori (Campylobacterales: Helicobacteraceae)

Abstract Helicobacter pylori (Marshall & Goodwin) is a widespread human pathogen that is acquiring resistance to the antibiotics used to treat it. This increasing resistance necessitates a continued search for new antibiotics. An antibiotic source that shows promise is animals whose immune systems must adapt to living in bacteria-laden conditions by producing antibacterial peptides or small molecules. Among these animals is the black soldier fly (BSF; Hermetia illucens Linnaeus), a Diptera that colonizes decomposing organic matter. In order to find anti-H. pylori peptides in BSF, larvae were challenged with Escherichia coli (Enterobacteriales: Enterobacteriaceae). Small peptides were extracted from hemolymph and purified using solid-phase extraction, molecular weight cutoff filtration and two rounds of preparative high performance liquid chromatography (HPLC). The anti-H. pylori fraction was followed through the purification process using the inhibition zone assay in brain-heart infusion agar, while peptides from uninoculated larvae had no activity. The inhibition halo of the active sample was comparable to the action of metronidazole in the inhibition zone assay. The purified sample contained four peptides with average masses of approximately 4.2 kDa that eluted together when analyzed by HPLC-mass spectrometry. The peptides likely have similar sequences, activity, and properties. Therefore, BSF produces inducible antibacterial peptides that have in vitro activity against H. pylori, which highlights BSF’s position as an important target for further bioprospecting.

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MICROBICIDAL POTENTIALITY OF PURIFIED ANTHOCYANIN FROM IN VITRO CULTURE OF CLERODENDRON INFORTUNATUM L. AGAINST SELECTED PATHOGENS

International Journal of Pharmacy and Pharmaceutical Sciences ◽

10.22159/ijpps.2018v10i6.18649 ◽

2018 ◽

Vol 10 (6) ◽

pp. 68

Author(s):

Meenu Krishnan V. G. ◽

Greeshma Murukan ◽

Aswathy J. M. ◽

Bosco Lawarence ◽

Murugan K.

Keyword(s):

Cell Suspension ◽

High Performance ◽

Solid Phase ◽

Membrane Integrity ◽

Inhibition Zone ◽

Molar Absorptivity ◽

Ms Medium ◽

Bacteria And Fungi ◽

Anthocyanin Production

Objective: Clerodendron infortunatum L. is a widely used medicinal herb over centuries for curing many skin-borne disorders. The present study was designed to validate the tribal knowledge by evaluating antimicrobial potential of purified anthocyanin extracted from in vitro cell suspension culture.Methods: The explants were inoculated on murashige and skoog (MS) medium mixed with various combinations of 2, 4-D a+BAP for callus induction. Green compact callus was initiated within 30 d from the explants on MS medium fortified with benzylaminopurine (BAP) (2.0 mg/l)+2, 4-D (0.5 mg/l). Subsequently, anthocyanin was triggered from the compact callus by subculturing in the medium containing 2, 4-D and Kinetin. Cell suspension culture was also developed. Anthocyanin production was enhanced by elicitation using salicylic acid and others. Three chromatographic methods such as solid phase extraction by Sepharose C18 column, Oasis-MCX and Amberlite XAD 7+Sephadex LH 120 sorbents were used to purify the in vitro synthesized anthocyanin from the cell cultures. HPLC and molar absorptivity assay were carried to check the purity. Antimicrobial analysis was also carried using standard protocols to check minimum inhibitory concentration (MIC) and minimum killing concentration (MKC).Results: The mean purity values obtained by high-performance thin layer chromatography (HPLC) were 90.9%±1.9, 80.60%±2.3 for Oasis MCX, Amberlite XAD-7+Sephadex LH-20 column respectively. However, the purity by molar absorptivity was found to be less. HPLC chromatogram revealed 12 fractions of anthocyanin. Inhibition zone diameter, MIC and MKC values obtained for the purified anthocyanin revealed its antimicrobial potentiality but at different levels among the selected bacteria and fungi. C. albicans, S. aureus, P. aerugenosa showed significant values followed by MRSA, E. coli and A. flavus. The results are comparable with the synthetic antibiotics. However, E. faecalis was more resistance. Mode of action was confirmed from the results of intracellular potassium leakage and bacterial membrane integrity analysis.Conclusion: Thus, the study confirms the efficacy of anthocyanin as natural antimicrobial and suggests the possibility of employing it as drugs for the treatment of infectious diseases caused by the pathogens.

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Chilean Rhubarb, Gunnera tinctoria (Molina) Mirb. (Gunneraceae): UHPLC-ESI-Orbitrap-MS Profiling of Aqueous Extract and its Anti-Helicobacter pylori Activity

Frontiers in Pharmacology ◽

10.3389/fphar.2020.583961 ◽

2021 ◽

Vol 11 ◽

Author(s):

Sonja Hebel-Gerber ◽

Apolinaria García-Cancino ◽

Angélica Urbina ◽

Mario J. Simirgiotis ◽

Javier Echeverría ◽

...

Keyword(s):

Helicobacter Pylori ◽

High Performance ◽

High Resolution Mass Spectrometry ◽

Partition Chromatography ◽

Centrifugal Partition Chromatography ◽

Electronic Microscopy ◽

Total Extract ◽

Transmission Electronic Microscopy ◽

H Pylori

The full UHPLC-MS metabolome fingerprinting and anti-Helicobacter pylori effect of Gunnera tinctoria (Molina) Mirb. (Nalca) total extract (GTE) and fractions prepared from its edible fresh petioles were evaluated. The activity of G. tinctoria against H. pylori strains ATCC 45504 and J99 was assessed in vitro by means of agar diffusion assay, Minimum Inhibition Concentration (MIC), and Minimum Bactericidal Concentration (MBC), while killing curve and transmission electronic microscopy (TEM) were conducted in order to determine the effect of the plant extract on bacterial growth and ultrastructure. Additionally, the inhibitory effect upon urease was evaluated using both the Jack Bean and H. pylori enzymes. To determine which molecules could be responsible for the antibacterial effects, tentative identification was done by ultra-high performance liquid chromatography coupled with high-resolution mass spectrometry (UHPLC-Q-Orbitrap®-HR-MS). Furthermore, the total G. tinctoria extract was fractionated using centrifugal partition chromatography (CPC), giving four active fractions (1–4). It was determined that the crude extract and centrifugal partition chromatography fractions of G. tinctoria have a bactericidal effect being the lowest MIC and MBC = 32 μg/ml. In the killing curves, fraction one acts faster than control amoxicillin. In the urease assay, F3 exhibited the lowest IC50 value of 13.5 μg/ml. Transmission electronic microscopy showed that crude G. tinctoria extract promotes disruption and separation of the cellular wall and outer membrane detachment on H. pylori causing bacterial cell death.

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The rod-to-coccoid body transformation in cultures of Helicobacter pylori

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100160686 ◽

1990 ◽

Vol 48 (3) ◽

pp. 626-627

Author(s):

A. R. Crooker ◽

W. G. Kraft ◽

T. L. Beard ◽

M. C. Myers

Keyword(s):

Helicobacter Pylori ◽

Growth Cycle ◽

Negative Bacterium ◽

Body Formation ◽

Coccoid Form ◽

Spiral Form ◽

Prolonged Culture ◽

H Pylori ◽

Upper Gastrointestinal

Helicobacter pylori is a microaerophilic, gram-negative bacterium found in the upper gastrointestinal tract of humans. There is strong evidence that H. pylori is important in the etiology of gastritis; the bacterium may also be a major predisposing cause of peptic ulceration. On the gastric mucosa, the organism exists as a spiral form with one to seven sheathed flagella at one (usually) or both poles. Short spirals were seen in the first successful culture of the organism in 1983. In 1984, Marshall and Warren reported a coccoid form in older cultures. Since that time, other workers have observed rod and coccal forms in vitro; coccoid forms predominate in cultures 3-7 days old. We sought to examine the growth cycle of H. pylori in prolonged culture and the mode of coccoid body formation.

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Synthesis and In Vitro Enzymatic Studies of New 3-Aryldiazenyl Indoles as Promising Helicobacter pylori IMPDH Inhibitors

Current Topics in Medicinal Chemistry ◽

10.2174/1568026619666190227212334 ◽

2019 ◽

Vol 19 (5) ◽

pp. 376-382 ◽

Cited By ~ 4

Author(s):

Sachin Jangra ◽

Gayathri Purushothaman ◽

Kapil Juvale ◽

Srimadhavi Ravi ◽

Aishwarya Menon ◽

...

Keyword(s):

Helicobacter Pylori ◽

Bacterial Infections ◽

Immunosuppressive Drugs ◽

Multi Drug Resistance ◽

Metabolic Enzyme ◽

World Population ◽

Inhibitor Molecule ◽

Nucleotide Synthesis ◽

H Pylori

Background & Objective:Helicobacter pylori infection is one of the primary causes of peptic ulcer followed by gastric cancer in the world population. Due to increased occurrences of multi-drug resistance to the currently available antibiotics, there is an urgent need for a new class of drugs against H. pylori. Inosine 5′-monophosphate dehydrogenase (IMPDH), a metabolic enzyme plays a significant role in cell proliferation and cell growth. It catalyses guanine nucleotide synthesis. IMPDH enzyme has been exploited as a target for antiviral, anticancer and immunosuppressive drugs. Recently, bacterial IMPDH has been studied as a potential target for treating bacterial infections. Differences in the structural and kinetic parameters of the eukaryotic and prokaryotic IMPDH make it possible to target bacterial enzyme selectively.Methods:In the current work, we have synthesised and studied the effect of substituted 3-aryldiazenyl indoles on Helicobacter pylori IMPDH (HpIMPDH) activity. The synthesised molecules were examined for their inhibitory potential against recombinant HpIMPDH.Results:In this study, compounds 1 and 2 were found to be the most potent inhibitors amongst the database with IC50 of 0.8 ± 0.02µM and 1 ± 0.03 µM, respectively.Conclusion:When compared to the most potent known HpIMPDH inhibitor molecule C91, 1 was only four-fold less potent and can be a good lead for further development of selective and potent inhibitors of HpIMPDH.

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Synthesis, Molecular Modelling and Antibacterial Activity Against Helicobacter pylori of Novel Diflunisal Derivatives as Urease Enzyme Inhibitors

Letters in Drug Design & Discovery ◽

10.2174/1570180815666180627130208 ◽

2019 ◽

Vol 16 (4) ◽

pp. 392-400 ◽

Cited By ~ 2

Author(s):

Göknil Pelin Coşkun ◽

Teodora Djikic ◽

Sadık Kalaycı ◽

Kemal Yelekçi ◽

Fikrettin Şahin ◽

...

Keyword(s):

Helicobacter Pylori ◽

Antibacterial Activity ◽

Enzyme Inhibitors ◽

Binding Modes ◽

Bacterial Strains ◽

Urease Enzyme ◽

H Pylori ◽

Ulcer Treatment ◽

Main Factor

Background:The main factor for the prolongation of the ulcer treatment in the gastrointestinal system would be Helicobacter pylori infection, which can possibly lead to gastrointestinal cancer. Triple therapy is the treatment of choice by today's standards. However, observed resistance among the bacterial strains can make the situation even worse. Therefore, there is a need to discover new targeted antibacterial therapy in order to make success in the eradication of H. pylori infections.Methods:The targeted therapy rule is to identify the related macromolecules that are responsible for the survival of the bacteria. Thus, 2-[(2',4'-difluoro-4-hydroxybiphenyl-3-yl)carbonyl]-N- (substituted)hydrazinocarbothioamide (3-13) and 5-(2',4'-difluoro-4-hydroxybiphenyl-3-yl)-4- (substituted)-2,4-dihydro-3H-1,2,4-triazole-3-thiones (14-17) were synthesized and evaluated for antibacterial activity in vitro against H. pylori.Results:All of the tested compounds showed remarkable antibacterial activity compared to the standard drugs (Ornidazole, Metronidazole, Nitrimidazin and Clarithromycin). Compounds 4 and 13 showed activity as 2µg/ml MIC value.Conclusion:In addition, we have investigated binding modes and energy of the compounds 4 and 13 on urease enzyme active by using the molecular docking tools.

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Nutrient Deficiency Promotes the Entry of Helicobacter pylori Cells into Candida Yeast Cells

Biology ◽

10.3390/biology10050426 ◽

2021 ◽

Vol 10 (5) ◽

pp. 426

Author(s):

Kimberly Sánchez-Alonzo ◽

Fabiola Silva-Mieres ◽

Luciano Arellano-Arriagada ◽

Cristian Parra-Sepúlveda ◽

Humberto Bernasconi ◽

...

Keyword(s):

Helicobacter Pylori ◽

Nutrient Deficiency ◽

Gastric Epithelium ◽

Yeast Cells ◽

Nutrient Concentrations ◽

Fetal Bovine ◽

H Pylori ◽

Pcr Techniques ◽

Strain Dependent

Helicobacter pylori, a Gram-negative bacterium, has as a natural niche the human gastric epithelium. This pathogen has been reported to enter into Candida yeast cells; however, factors triggering this endosymbiotic relationship remain unknown. The aim of this work was to evaluate in vitro if variations in nutrient concentration in the cultured medium trigger the internalization of H. pylori within Candida cells. We used H. pylori–Candida co-cultures in Brucella broth supplemented with 1%, 5% or 20% fetal bovine serum or in saline solution. Intra-yeast bacteria-like bodies (BLBs) were observed using optical microscopy, while intra-yeast BLBs were identified as H. pylori using FISH and PCR techniques. Intra-yeast H. pylori (BLBs) viability was confirmed using the LIVE/DEAD BacLight Bacterial Viability kit. Intra-yeast H. pylori was present in all combinations of bacteria–yeast strains co-cultured. However, the percentages of yeast cells harboring bacteria (Y-BLBs) varied according to nutrient concentrations and also were strain-dependent. In conclusion, reduced nutrients stresses H. pylori, promoting its entry into Candida cells. The starvation of both H. pylori and Candida strains reduced the percentages of Y-BLBs, suggesting that starving yeast cells may be less capable of harboring stressed H. pylori cells. Moreover, the endosymbiotic relationship between H. pylori and Candida is dependent on the strains co-cultured.

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Putative Cooperative ATP–DnaA Binding to Double-Stranded DnaA Box and Single-Stranded DnaA-Trio Motif upon Helicobacter pylori Replication Initiation Complex Assembly

International Journal of Molecular Sciences ◽

10.3390/ijms22126643 ◽

2021 ◽

Vol 22 (12) ◽

pp. 6643

Author(s):

Pawel Jaworski ◽

Dorota Zyla-Uklejewicz ◽

Malgorzata Nowaczyk-Cieszewska ◽

Rafal Donczew ◽

Thorsten Mielke ◽

...

Keyword(s):

Helicobacter Pylori ◽

Replication Initiation ◽

Dna Unwinding ◽

Rich Region ◽

Initiator Protein ◽

New Class ◽

H Pylori ◽

General Architecture ◽

Dnaa Box

oriC is a region of the bacterial chromosome at which the initiator protein DnaA interacts with specific sequences, leading to DNA unwinding and the initiation of chromosome replication. The general architecture of oriCs is universal; however, the structure of oriC and the mode of orisome assembly differ in distantly related bacteria. In this work, we characterized oriC of Helicobacter pylori, which consists of two DnaA box clusters and a DNA unwinding element (DUE); the latter can be subdivided into a GC-rich region, a DnaA-trio and an AT-rich region. We show that the DnaA-trio submodule is crucial for DNA unwinding, possibly because it enables proper DnaA oligomerization on ssDNA. However, we also observed the reverse effect: DNA unwinding, enabling subsequent DnaA–ssDNA oligomer formation—stabilized DnaA binding to box ts1. This suggests the interplay between DnaA binding to ssDNA and dsDNA upon DNA unwinding. Further investigation of the ts1 DnaA box revealed that this box, together with the newly identified c-ATP DnaA box in oriC1, constitute a new class of ATP–DnaA boxes. Indeed, in vitro ATP–DnaA unwinds H. pylori oriC more efficiently than ADP–DnaA. Our results expand the understanding of H. pylori orisome formation, indicating another regulatory pathway of H. pylori orisome assembly.

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Shedding Light on the African Enigma: In Vitro Testing of Homo sapiens-Helicobacter pylori Coevolution

Microorganisms ◽

10.3390/microorganisms9020240 ◽

2021 ◽

Vol 9 (2) ◽

pp. 240

Author(s):

Bruno Cavadas ◽

Marina Leite ◽

Nicole Pedro ◽

Ana C. Magalhães ◽

Joana Melo ◽

...

Keyword(s):

Gene Expression ◽

Helicobacter Pylori ◽

Host Response ◽

Homo Sapiens ◽

European Ancestry ◽

Genome Wide ◽

Expression Host ◽

H Pylori ◽

Human Ancestry

The continuous characterization of genome-wide diversity in population and case–cohort samples, allied to the development of new algorithms, are shedding light on host ancestry impact and selection events on various infectious diseases. Especially interesting are the long-standing associations between humans and certain bacteria, such as the case of Helicobacter pylori, which could have been strong drivers of adaptation leading to coevolution. Some evidence on admixed gastric cancer cohorts have been suggested as supporting Homo-Helicobacter coevolution, but reliable experimental data that control both the bacterium and the host ancestries are lacking. Here, we conducted the first in vitro coinfection assays with dual human- and bacterium-matched and -mismatched ancestries, in African and European backgrounds, to evaluate the genome wide gene expression host response to H. pylori. Our results showed that: (1) the host response to H. pylori infection was greatly shaped by the human ancestry, with variability on innate immune system and metabolism; (2) African human ancestry showed signs of coevolution with H. pylori while European ancestry appeared to be maladapted; and (3) mismatched ancestry did not seem to be an important differentiator of gene expression at the initial stages of infection as assayed here.

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A High-Throughput Metabolic Microarray Assay Reveals Antibacterial Effects of Black and Red Raspberries and Blackberries against Helicobacter pylori Infection

Antibiotics ◽

10.3390/antibiotics10070845 ◽

2021 ◽

Vol 10 (7) ◽

pp. 845

Author(s):

Candace Goodman ◽

Katrina N. Lyon ◽

Aitana Scotto ◽

Cyra Smith ◽

Thomas A. Sebrell ◽

...

Keyword(s):

Helicobacter Pylori ◽

High Throughput ◽

Antibacterial Properties ◽

Treatment Strategies ◽

Helicobacter Pylori Infection ◽

Antibacterial Effects ◽

Biolog System ◽

Freeze Dried ◽

H Pylori

Helicobacter pylori infection is commonly treated with a combination of antibiotics and proton pump inhibitors. However, since H. pylori is becoming increasingly resistant to standard antibiotic regimens, novel treatment strategies are needed. Previous studies have demonstrated that black and red berries may have antibacterial properties. Therefore, we analyzed the antibacterial effects of black and red raspberries and blackberries on H. pylori. Freeze-dried powders and organic extracts from black and red raspberries and blackberries were prepared, and high-performance liquid chromatography was used to measure the concentrations of anthocyanins, which are considered the major active ingredients. To monitor antibiotic effects of the berry preparations on H. pylori, a high-throughput metabolic growth assay based on the Biolog system was developed and validated with the antibiotic metronidazole. Biocompatibility was analyzed using human gastric organoids. All berry preparations tested had significant bactericidal effects in vitro, with MIC90 values ranging from 0.49 to 4.17%. Antimicrobial activity was higher for extracts than powders and appeared to be independent of the anthocyanin concentration. Importantly, human gastric epithelial cell viability was not negatively impacted by black raspberry extract applied at the concentration required for complete bacterial growth inhibition. Our data suggest that black and red raspberry and blackberry extracts may have potential applications in the treatment and prevention of H. pylori infection but differ widely in their MICs. Moreover, we demonstrate that the Biolog metabolic assay is suitable for high-throughput antimicrobial susceptibility screening of H. pylori.

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In Vitro Incorporation of Helicobacter pylori into Candida albicans Caused by Acidic pH Stress

Pathogens ◽

10.3390/pathogens9060489 ◽

2020 ◽

Vol 9 (6) ◽

pp. 489 ◽

Cited By ~ 1

Author(s):

Kimberly Sánchez-Alonzo ◽

Cristian Parra-Sepúlveda ◽

Samuel Vega ◽

Humberto Bernasconi ◽

Víctor L. Campos ◽

...

Keyword(s):

Helicobacter Pylori ◽

Candida Albicans ◽

Pcr Amplification ◽

Growth Curves ◽

Acidic Ph ◽

Ph Values ◽

16S Gene ◽

Wide Range ◽

H Pylori

Yeasts can adapt to a wide range of pH fluctuations (2 to 10), while Helicobacter pylori, a facultative intracellular bacterium, can adapt to a range from pH 6 to 8. This work analyzed if H. pylori J99 can protect itself from acidic pH by entering into Candida albicans ATCC 90028. Growth curves were determined for H. pylori and C. albicans at pH 3, 4, and 7. Both microorganisms were co-incubated at the same pH values, and the presence of intra-yeast bacteria was evaluated. Intra-yeast bacteria-like bodies were detected using wet mounting, and intra-yeast binding of anti-H. pylori antibodies was detected using immunofluorescence. The presence of the H. pylori rDNA 16S gene in total DNA from yeasts was demonstrated after PCR amplification. H. pylori showed larger death percentages at pH 3 and 4 than at pH 7. On the contrary, the viability of the yeast was not affected by any of the pHs evaluated. H. pylori entered into C. albicans at all the pH values assayed but to a greater extent at unfavorable pH values (pH 3 or 4, p = 0.014 and p = 0.001, respectively). In conclusion, it is possible to suggest that H. pylori can shelter itself within C. albicans under unfavorable pH conditions.

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