Pharmacodynamics of Moxifloxacin, Meropenem, Caspofungin and their Combinations Against In Vitro Polymicrobial Inter-kingdom Biofilms

2021 ◽  
Author(s):  
Albert Ruiz-Sorribas ◽  
Hervé Poilvache ◽  
Françoise Van Bambeke
Keyword(s):  
Confocal Microscopy ◽  
Medical Devices ◽  
Drug Response ◽  
Single Agent ◽  
Total Biomass ◽  
Additive Effects ◽  
E Coli ◽  
Biofilm Model ◽  
Microscopy Images

Biofilms colonize medical devices and are often recalcitrant to antibiotics. Inter-kingdom biofilms, when at least a bacterium and a fungus are co-isolated, increase the likelihood of therapeutic failures. In this work, a three-species in vitro biofilm model including S. aureus , E. coli and C. albicans was used to study the activity of the antibiotics moxifloxacin and meropenem, the antifungal caspofungin, and combinations of them against inter-kingdom biofilms. The culturable cells and total biomass were evaluated to determine the pharmacodynamic parameters of the drug response for the incubation with the drugs alone. The synergic or antagonistic effects (increased/decreased effects) of the combination of drugs were analysed with the highest single agent method. Biofilms were imaged in confocal microscopy after live/dead staining. The drugs had limited activity when used alone against single-, dual- or three-species biofilms. When used in combination, additive effects were observed against single- or dual-species biofilms, and increased effects (synergy) against biomass of three-species biofilms. In addition, the two antibiotics showed different patterns, moxifloxacin being more active when targeting S. aureus and meropenem when targeting E. coli . All these observations were confirmed by confocal microscopy images. Our findings highlight the interest in combining caspofungin with antibiotics against inter-kingdom biofilms.

scholarly journals Metabolic Modeling of Clostridium difficile Associated Dysbiosis of the Gut Microbiota

Processes ◽  
2019 ◽  
Vol 7 (2) ◽  
pp. 97 ◽  
Author(s):  
Poonam Phalak ◽  
Michael Henson
Keyword(s):  
Clostridium Difficile ◽  
Treatment Strategies ◽  
E Coli ◽  
Biofilm Model ◽  
Commensal Species ◽  
Species Abundances ◽  
New Treatment ◽  
Biofilm Development

Recent in vitro experiments have demonstrated the ability of the pathogen Clostridium difficile and commensal gut bacteria to form biofilms on surfaces, and biofilm development in vivo is likely. Various studies have reported that 3%–15% of healthy adults are asymptomatically colonized with C. difficile, with commensal species providing resistance against C. difficile pathogenic colonization. C. difficile infection (CDI) is observed at a higher rate in immunocompromised patients previously treated with broad spectrum antibiotics that disrupt the commensal microbiota and reduce competition for available nutrients, resulting in imbalance among commensal species and dysbiosis conducive to C. difficile propagation. To investigate the metabolic interactions of C. difficile with commensal species from the three dominant phyla in the human gut, we developed a multispecies biofilm model by combining genome-scale metabolic reconstructions of C. difficile, Bacteroides thetaiotaomicron from the phylum Bacteroidetes, Faecalibacterium prausnitzii from the phylum Firmicutes, and Escherichia coli from the phylum Proteobacteria. The biofilm model was used to identify gut nutrient conditions that resulted in C. difficile-associated dysbiosis characterized by large increases in C. difficile and E. coli abundances and large decreases in F. prausnitzii abundance. We tuned the model to produce species abundances and short-chain fatty acid levels consistent with available data for healthy individuals. The model predicted that experimentally-observed host-microbiota perturbations resulting in decreased carbohydrate/increased amino acid levels and/or increased primary bile acid levels would induce large increases in C. difficile abundance and decreases in F. prausnitzii abundance. By adding the experimentally-observed perturbation of increased host nitrate secretion, the model also was able to predict increased E. coli abundance associated with C. difficile dysbiosis. In addition to rationalizing known connections between nutrient levels and disease progression, the model generated hypotheses for future testing and has the capability to support the development of new treatment strategies for C. difficile gut infections.

scholarly journals In vitro observation: the GFP-E. coli adhering to porcine erythrocytes can be removed by porcine alveolar macrophages

PeerJ ◽  
2019 ◽  
Vol 7 ◽  
pp. e6439 ◽  
Author(s):  
Wei Yin ◽  
Chun Wang ◽  
Kuohai Fan ◽  
Na Sun ◽  
Yaogui Sun ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Confocal Microscopy ◽  
Fluorescence Intensity ◽  
Alveolar Macrophages ◽  
Control Group ◽  
Laser Confocal Microscopy ◽  
Functional Protein ◽  
Blank Control Group ◽  
E Coli

Although the activation of pathogen phagocytosis via complement system has been studied, erythrocyte-phagocyte interactions in pigs are not clearly understood. Therefore, we sought to investigate the ability of porcine erythrocytes to clear immune complexes (ICs) by using laser confocal microscopy and flow cytometry to observe the immune adhesion of porcine erythrocytes to fluorescent bacilli and the immune presentation process of transferring fluorescent bacilli to macrophages. Isolated porcine alveolar macrophages (PAMs) had uniform morphology and size, and a survival rate of 97.2%. The phagocytosis rate was 98.8%. After WT E. coli was labeled with Fluorescein Isothiocyanate (FITC), the bacteria showed a bright green fluorescence, and the labeling rate was 92.3%. When laser confocal microscopy was utilized to observe the co-incubation system of porcine erythrocytes, PAM, and fluorescent E. coli, the fluorescence intensity of bacilli decreased with increasing observation time and even disappeared. Flow Cytometry examination showed that the average fluorescence intensity of PAMs co-incubated with porcine erythrocytes adhered to WT-E. coli-FITC, was significantly higher than that of normal PAMs. Furthermore, when porcine erythrocytes adhered to WT E. coli were incubated with PAMs, the surface mean fluorescence intensity of porcine erythrocytes was significantly higher than that of the blank control group. This shows that PAMs can competitively bind to the oposinized E. coli adhered to the surface of porcine erythrocytes, and these oposinized pathogens can enter macrophages by the process of phagocytosis, which promoting the internalization of ICs or pathogens. During this process, the physical morphology of porcine erythrocytes was not damaged, but the levels of its main functional protein CR1-like were reduced.

scholarly journals Inhibitors of Bcl-2 and Bruton’s tyrosine kinase synergize to abrogate diffuse large B-cell lymphoma growth in vitro and in orthotopic xenotransplantation models

Leukemia ◽  
2021 ◽  
Author(s):  
Katrin Bertram ◽  
Peter John Leary ◽  
Christophe Boudesco ◽  
Jonas Fullin ◽  
Kristin Stirm ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
B Cell ◽  
Drug Response ◽  
Cell Lymphoma ◽  
Single Agent ◽  
Acquired Resistance ◽  
B Cell Lymphoma ◽  
Large B Cell Lymphoma ◽  
Large B Cell

AbstractNumerous targeted therapies have been developed for diffuse large B-cell lymphoma, but the results of late-stage clinical trials were mostly disappointing and have led to very few new regulatory approvals. Here, we use single and combinatorial drug response profiling to show that the combined inhibition of the anti-apoptotic protein Bcl-2 and of the tyrosine kinase BTK with the small molecules venetoclax and ibrutinib efficiently kills DLBCL cells in vitro. High Bcl-2 expression due to either BCL2 amplifications or translocations, in conjunction with chronic active BCR signaling accurately predict responses to dual Bcl-2/BTK inhibition. Orthotopic xenotransplantation and patient-derived xenograft models confirm that the combinatorial is superior to single-agent treatment in reducing the lymphoma burden. Combinatorial treatment further efficiently overcomes both primary and acquired resistance to venetoclax, which we could link to reduced expression of the Bcl-2 family members Bcl-XL and Bcl-2A1 under ibrutinib. We found in a Swiss DLBCL cohort that ~15% of patients are projected to respond to the venetoclax/ibrutinib combination based on their high Bcl-2 expression and nuclear NF-κB localization. Our data show that drug sensitivities exposed by drug response profiling can be attributed to specific mutational signatures and immunohistochemical biomarkers, and point to combined Bcl-2/BTK inhibition as a promising therapeutic strategy in DLBCL.

scholarly journals The Glutaminase Activity of L-Asparaginase Mediates Suppression of Asns Upregulation

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 3959-3959 ◽  
Author(s):  
Wai Kin Chan ◽  
Lin Tan ◽  
Karine Harutyunyan ◽  
Di Du ◽  
Leona Martin ◽  
...  
Keyword(s):  
Cell Death ◽  
Amino Acid ◽  
Anticancer Activity ◽  
Single Agent ◽  
Leukemia Cell ◽  
Cell Types ◽  
Leukemia Cells ◽  
E Coli ◽  
Glutaminase Activity

Abstract L-asparaginase (ASNase) is a standard component of treatment regimens used for acute lymphoblastic leukemia and is being tested against other cancer types, including acute myeloid leukemia, lymphoma, and pancreatic cancer. We and others have reported that the anticancer activity of ASNase requires the enzyme's glutaminase activity, but the underlying glutaminase-mediated mechanism(s) that lead to leukemia cell death are unknown. Glutamine, the most abundant amino acid in the blood, is known for pleiotropic roles in numerous biological pathways, including energy metabolism, redox metabolism, nucleotide anabolism, and amino acid anabolism. Many cancer cells have been found to reprogram their metabolic pathways to become highly dependent on glutamine for survival and proliferation. Glutaminase (GLS/GLS2)-mediated conversion of glutamine to glutamic acid provides the latter as a substrate for conversion to α‐ketoglutarate by transaminases or glutamate dehydrogenases (GLUD1/GLUD2) to fuel the TCA cycle. Consequently, targeting glutamine metabolism has become an attractive strategy for anticancer therapy. The enzyme asparagine synthetase (ASNS) mediates resistance to ASNase through synthesis of asparagine. ASNS is expressed in most cell types, and its expression is upregulated in response to a wide variety of cell stresses, including amino acid limitation and endoplasmic reticulum stress. We and others have shown that ASNS-positive leukemia cells capable of synthesizing asparagine de novo are less responsive than ASNS-negative leukemia cells to ASNase therapy (Chan et al., Blood, 2014). Moreover, ASNase resistance has been associated with elevated ASNS expression. In fact, we have shown that ASNS expression is a predictive marker of the in vitro response of leukemia cell lines and some solid tumor cell types to ASNase. The expression of ASNS in most cells in the body poses a serious challenge for successful therapy with ASNase; for example, production of asparagine by the liver and cells (e.g., mesenchymal stem cells and adipocytes) of the tumor microenvironment may contribute significantly to ASNase resistance in vivo. Here we used the high-glutaminase E. chrysanthemi ASNase (Erwinaze®), wild-type E. coli ASNase (ASNaseWT), and the glutaminase-deficient E. coli mutant, ASNaseQ59L, as models of high, medium-, and low-glutaminase, respectively, to explore ASNase glutaminase activity-mediated mechanisms of leukemia cell death. Unexpectedly, we found that increasing glutaminase activity caused an increase in the suppression of ASNS upregulation in vitro (Figure 1A). In NSG mice injected with luciferase-labeled Sup-B15 cells, single-agent ASNaseWT yielded a durable response approximating cure, whereas glutaminase-deficient ASNaseQ59L yielded a complete response but with recurrence. Together, the results suggest that ASNase glutaminase activity is associated with suppression of ASNS upregulation, making durable, single-agent anticancer activity easier to achieve. Overall, the results provide new insight into the mechanism of action of ASNase. Disclosures Konopleva: Stemline Therapeutics: Research Funding. Weinstein:NIH: Patents & Royalties: L-asparaginase. Lorenzi:Erytech Pharma: Consultancy; NIH: Patents & Royalties.

scholarly journals The potential for developing new antimicrobial resistance from the use of medical devices containing chlorhexidine, minocycline, rifampicin and their combinations: a systematic review

2020 ◽  
Vol 2 (1) ◽  
Author(s):  
Ruth A Reitzel ◽  
Joel Rosenblatt ◽  
Bahgat Z Gerges ◽  
Andrew Jarjour ◽  
Ana Fernández-Cruz ◽  
...  
Keyword(s):  
Systematic Review ◽  
Antimicrobial Resistance ◽  
Medical Devices ◽  
Single Agent ◽  
Resistance Selection ◽  
Induce Resistance ◽  
Study Type ◽  
Catheter Infections

Abstract Background Catheter infections remain one of the most persistent adverse events causing significant morbidity, economic impact and mortality. Several strategies have been proposed to reduce these infections including the use of catheters embedded with antibiotics and/or antiseptics. One reoccurring challenge is the fear that antimicrobial medical devices will induce resistance. The aim of this systematic review is to evaluate the evidence for induced antimicrobial resistance caused by exposure to antimicrobial medical devices. Methods Four electronic databases [MEDLINE, Embase, Cumulative Index to Nursing and Allied Health Literature (CINAHL) and Scopus] were screened for studies published between 1983 and 2019 regarding assessment of microbial resistance with use of medical devices containing chlorhexidine, minocycline, rifampicin or combinations thereof. Development of new resistance, selection for tolerant organisms and ‘no change in resistance’ were assessed. Results Forty-four publications, grouped by study type and stratified by drug assessed, were included for analyses. The majority of studies found no change in resistance after exposure to antimicrobial medical devices (13 in vitro, 2 in vivo, 20 clinical). Development of new resistance was commonly reported with the use of rifampicin as a single agent and only reported in one study assessing the minocycline/rifampicin combination (M/R); however, the increase in MIC was well below clinical relevance. Conclusions Emergence of new resistance to combinations of M/R, minocycline/rifampicin/chlorhexidine (M/R/CH) and chlorhexidine/silver sulfadiazine (CHXSS) was rare. No clinical trials confirmed its occurrence and some refuted it. The risk of development of new resistance to these antimicrobial combinations appears more fear-based than substantiated by clinical and experimental evidence but warrants continued surveillance.

Rapid, Refined, and Robust Method for Expression, Purification, and Characterization of Recombinant Human Amyloid-beta M1-42

2019 ◽  
Author(s):  
Priya Prakash ◽  
Travis Lantz ◽  
Krupal P. Jethava ◽  
Gaurav Chopra
Keyword(s):  
Amyloid Beta ◽  
Western Blots ◽  
Force Microscopy ◽  
E Coli ◽  
Atomic Force ◽  
Set Up ◽  
Short Time

Amyloid plaques found in the brains of Alzheimer’s disease (AD) patients primarily consists of amyloid beta 1-42 (Ab42). Commercially, Ab42 is synthetized using peptide synthesizers. We describe a robust methodology for expression of recombinant human Ab(M1-42) in Rosetta(DE3)pLysS and BL21(DE3)pLysS competent E. coli with refined and rapid analytical purification techniques. The peptide is isolated and purified from the transformed cells using an optimized set-up for reverse-phase HPLC protocol, using commonly available C18 columns, yielding high amounts of peptide (~15-20 mg per 1 L culture) in a short time. The recombinant Ab(M1-42) forms characteristic aggregates similar to synthetic Ab42 aggregates as verified by western blots and atomic force microscopy to warrant future biological use. Our rapid, refined, and robust technique to purify human Ab(M1-42) can be used to synthesize chemical probes for several downstream in vitro and in vivo assays to facilitate AD research.

Антимикробная активность лиофилизированного водного извлечения Caragana jubata (Pall.) Poir.

2020 ◽  
Vol 54 (3) ◽  
pp. 42-44
Author(s):  
Павел Алексеевич Какорин ◽  
Татьяна Владимировна Фатеева ◽  
Ольга Ивановна Терешкина ◽  
Ирина Борисовна Перова ◽  
Галина Владиславовна Раменская ◽  
...  
Keyword(s):  
E Coli

На основании ранее проведенных исследований установлен профиль флавоноидов лиофилизированного водного извлечения, полученного из побегов C. jubata. В связи с тем, что, согласно данным литературы, флавоноиды являются потенциальными ингибиторами микроорганизмов, проведено изучение антимикробной активности лиофилизата в опытах in vitro с использованием скринигового метода определения антимикробной активности для препаратов растительного происхождения. При изучении бактериостатической и фунгистатической активности в опытах in vitro использовали метод двукратного серийного разведения препаратов в жидких питательных средах. В результате исследования лиофилизированного водного извлечения караганы гривастой установлено наличие умеренной антимикробной активности в отношении всех изученных штаммов патогенных микроорганизмов: грамположительных и грамотрицательных бактерий (S. aureus, E. coli, P. vulgaris, P. aeruginosa), дрожжеподобных и мицелиальных грибов (C. albicans, M. canis). Полученные данные позволяют рекомендовать лиофилизированное водное извлечение караганы гривастой для создания на его основе лекарственных форм наружного применения для лечения заболеваний кожи и слизистых оболочек, связанных с бактериальным воспалительным процессом.

Влияние in vitro изолированного и сочетанного с антибактериальными средствами применения бовгиалуронидазы азоксимер на целостность бактериальной биопленки и жизнеспосоность микроорганизмов

2020 ◽  
Vol 83 (2) ◽  
pp. 38-44
Author(s):  
Е. Ю. Тризна ◽  
Д. Р. Байдамшина ◽  
Александр А. Виницкий ◽  
А. Р. Каюмов
Keyword(s):  
E Coli

Исследована способность лиофилизата бовгиалуронидазы азоксимера («Лонгидаза») разрушать бактериальные биопленки S. aureus, E. faecalis, E. coli, а также сочетанное действие препарата с антибактериальными средствами. Показано, что 2 ч инкубации бовгиалуронидазы азоксимер в концентрации 750 – 1500 МЕ/мл вызывает двукратное снижение биомассы матрикса зрелых биопленок E. faecalis и E. coli, и на 60 % — S. aureus. Данный ферментный препарат не влияет на образование бактериальных биопленок. При сочетанном применении с антибактериальными средствами препарат повышает их эффективность в отношении бактерий в составе биопленок. Так, концентрация ципро-флоксацина и амоксициллина, необходимая для снижения количества КОЕ на 3 порядка в биопленке E. faecalis, в присутствии бовгиалуронидазы азоксимера снижается в 16 раз (p < 0,05). В присутствии фермента в 16 раз меньшие концентрации цефуроксима, фосфомицина, ципрофлоксацина и амикацина достаточны для снижения количества КОЕ на 3 порядка в биопленке E. coli (p < 0,05), и в значительно меньшей концентрации цефуроксим оказывает бактерицидное действие на клетки в биопленке S. aureus (p < 0,05). Вероятно, бовгиалуронидаза азоксимер увеличивает проникновение антибактериальных средств к клеткам бактерий в биопленке, что обеспечивает потенцирование их антибактериального эффекта. Такое действие ферментного препарата позволяет снизить дозу и повысить безопасность антибактериальных средств при сохранении их эффективности.

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