scholarly journals Highly parallelized droplet cultivation and prioritization of antibiotic producers from natural microbial communities

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Lisa Mahler ◽  
Sarah P Niehs ◽  
Karin Martin ◽  
Thomas Weber ◽  
Kirstin Scherlach ◽  
...  
Keyword(s):  
Stochastic Process ◽  
Antimicrobial Activity ◽  
High Throughput Screening ◽  
Single Cells ◽  
Agar Plate ◽  
Antimicrobial Compounds ◽  
Cultivation System ◽  
Strain Isolation ◽  
System A ◽  
Reporter Strains

Antibiotics from few culturable microorganisms have saved millions of lives since the 20th century. But with resistance formation, these compounds become increasingly ineffective, while the majority of microbial and with that chemical compound diversity remains inaccessible for cultivation and exploration. Culturing recalcitrant bacteria is a stochastic process. But conventional methods are limited to low throughput. By increasing (i) throughput and (ii) sensitivity by miniaturization, we innovate microbiological cultivation to comply with biological stochasticity. Here, we introduce a droplet-based microscale cultivation system, which is directly coupled to a high-throughput screening for antimicrobial activity prior to strain isolation. We demonstrate that highly parallelized in-droplet cultivation starting from single cells results in the cultivation of yet uncultured species and a significantly higher bacterial diversity than standard agar plate cultivation. Strains able to inhibit intact reporter strains were isolated from the system. A variety of antimicrobial compounds were detected for a selected potent antibiotic producer.

scholarly journals Highly parallelized microfluidic droplet cultivation and prioritization on antibiotic producers from complex natural microbial communities

2019 ◽  
Author(s):  
Lisa Mahler ◽  
Sarah Niehs ◽  
Karin Martin ◽  
Thomas Weber ◽  
Kirstin Scherlach ◽  
...  
Keyword(s):  
Antimicrobial Activity ◽  
Natural Products ◽  
Bacterial Diversity ◽  
High Throughput ◽  
High Throughput Screening ◽  
Single Cells ◽  
Amplicon Sequencing ◽  
Droplet Microfluidics ◽  
Rrna Gene ◽  
Bacterial Strains

AbstractTo investigate the overwhelming part of the bacterial diversity still evading standard cultivation for its potential use in antibiotic synthesis, we have compiled a microscale-cultivation and screening system. We devised a strategy based on droplet-microfluidics taking advantage of the inherent miniaturization and high throughput. Single cells of natural samples were confined in 9 x 106 aqueous droplets and subjected to long-term incubation under controlled conditions. Subsequent a high-throughput screening for antimicrobial natural products was implemented, employing a whole cell reporting system using the viability of reporter strains as a probe for antimicrobial activity. Due to the described microscale cultivation a novel subset of bacterial strains was made available for the following screening for antimicrobials. We demonstrate the merits of the in-droplet cultivation by comparing the cultivation outcome in microfluidic droplets and on conventional agar plates for a bacterial community derived from soil by 16S rRNA gene amplicon sequencing. In-droplet cultivation resulted in a significantly higher bacterial diversity without the common overrepresentation of Firmicutes. Natural strains able to inhibit either a Gram-positive or a Gram-negative reporter strain were isolated from the microscale system and further cultivated. Thereby a variety of rare isolates was obtained. The natural products with antimicrobial activity were elucidated for the most promising candidate. Our method combines a new cultivation approach with a high-throughput search for antibiotic producers to increase the chances of finding new lead substances.

ANTIMICROBIAL ACTIVITIES OF METHANOL EXTRACT FROM UNIDENTIFIED SPONGE ASSOCIATED BACTERIA IN LEMUKUTAN ISLAND, KALIMANTAN BARAT

2009 ◽  
Vol 1 (2) ◽  
Author(s):  
Risa Nofiani ◽  
Siti Nurbetty ◽  
Ajuk Sapar
Keyword(s):  
Antimicrobial Activity ◽  
Bacillus Subtilis ◽  
Pathogenic Bacteria ◽  
Methanol Extract ◽  
Agar Medium ◽  
Antimicrobial Activities ◽  
Antimicrobial Compounds ◽  
Associated Bacteria ◽  
Minimum Inhibitory Concentrations ◽  
Sponge Associated Bacteria

<p>The increase of issues on the antibiotics resistant pathogenic bacteria has triggered high exploration for new antimicrobial compounds. One of the potential sources is sponge-associated bacteria. The aim of this study was to get sponge-associated bacteria extract containing antimicrobial activities. On the basis screening of antimicrobial activity using by streaking on agar medium, there were two potential isolates with antimicrobial activities namely LCS1 and LCS2. The two isolates were cultivated,then secondary metabolite product were extracted using methanol as a solvent. Minimum inhibitory concentrations (MICs) of extract LCS 1 were 1,000 μg/well for S. aureus, 950 μg/well for Salmonella sp.and 800 μg/well for Bacillus subtilis. Minimum inhibitory concentrations of extract LCS 2 were 500 μg/well for S. aureus, 1,050 μg/well for Salmonella sp., 750 μg/well for Bacillus subtilis, 350 μg/well for P. aeruginosa, 750 μg/sumur terhadap B. subtilis. Based on the MIC values, the two assay extracts have a relatively low antimicrobial activity.</p> <p>Keywords:Antimicrobial,Sponges associated bacteria,MICs</p>

scholarly journals High-throughput screening and rational design of biofunctionalized surfaces with optimized biocompatibility and antimicrobial activity

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Zhou Fang ◽  
Junjian Chen ◽  
Ye Zhu ◽  
Guansong Hu ◽  
Haoqian Xin ◽  
...  
Keyword(s):  
Antimicrobial Activity ◽  
High Throughput ◽  
High Throughput Screening ◽  
Rational Design ◽  
Click Reaction ◽  
Reaction Times ◽  
Great Promise ◽  
Biomaterial Surfaces

AbstractPeptides are widely used for surface modification to develop improved implants, such as cell adhesion RGD peptide and antimicrobial peptide (AMP). However, it is a daunting challenge to identify an optimized condition with the two peptides showing their intended activities and the parameters for reaching such a condition. Herein, we develop a high-throughput strategy, preparing titanium (Ti) surfaces with a gradient in peptide density by click reaction as a platform, to screen the positions with desired functions. Such positions are corresponding to optimized molecular parameters (peptide densities/ratios) and associated preparation parameters (reaction times/reactant concentrations). These parameters are then extracted to prepare nongradient mono- and dual-peptide functionalized Ti surfaces with desired biocompatibility or/and antimicrobial activity in vitro and in vivo. We also demonstrate this strategy could be extended to other materials. Here, we show that the high-throughput versatile strategy holds great promise for rational design and preparation of functional biomaterial surfaces.

scholarly journals Comparison of a Short Linear Antimicrobial Peptide with Its Disulfide-Cyclized and Cyclotide-Grafted Variants against Clinically Relevant Pathogens

2021 ◽  
Vol 9 (6) ◽  
pp. 1249
Author(s):  
Johannes Koehbach ◽  
Jurnorain Gani ◽  
Kai Hilpert ◽  
David J Craik
Keyword(s):  
Antimicrobial Activity ◽  
Antimicrobial Peptide ◽  
Human Serum ◽  
World Health ◽  
Resistant Bacteria ◽  
Moderate Activity ◽  
Antimicrobial Compounds ◽  
Strong Activity ◽  
Linear Peptide ◽  
Linear Version

According to the World Health Organization (WHO) the development of resistance against antibiotics by microbes is one of the most pressing health concerns. The situation will intensify since only a few pharmacological companies are currently developing novel antimicrobial compounds. Discovery and development of novel antimicrobial compounds with new modes of action are urgently needed. Antimicrobial peptides (AMPs) are known to be able to kill multidrug-resistant bacteria and, therefore, of interest to be developed into antimicrobial drugs. Proteolytic stability and toxicities of these peptides are challenges to overcome, and one strategy frequently used to address stability is cyclization. Here we introduced a disulfide-bond to cyclize a potent and nontoxic 9mer peptide and, in addition, as a proof-of-concept study, grafted this peptide into loop 6 of the cyclotide MCoTI-II. This is the first time an antimicrobial peptide has been successfully grafted onto the cyclotide scaffold. The disulfide-cyclized and grafted cyclotide showed moderate activity in broth and strong activity in 1/5 broth against clinically relevant resistant pathogens. The linear peptide showed superior activity in both conditions. The half-life time in 100% human serum was determined, for the linear peptide, to be 13 min, for the simple disulfide-cyclized peptide, 9 min, and, for the grafted cyclotide 7 h 15 min. The addition of 10% human serum led to a loss of antimicrobial activity for the different organisms, ranging from 1 to >8-fold for the cyclotide. For the disulfide-cyclized version and the linear version, activity also dropped to different degrees, 2 to 18-fold, and 1 to 30-fold respectively. Despite the massive difference in stability, the linear peptide still showed superior antimicrobial activity. The cyclotide and the disulfide-cyclized version demonstrated a slower bactericidal effect than the linear version. All three peptides were stable at high and low pH, and had very low hemolytic and cytotoxic activity.

scholarly journals Highlighting the Biotechnological Potential of Deep Oceanic Crust Fungi through the Prism of Their Antimicrobial Activity

Marine Drugs ◽  
2021 ◽  
Vol 19 (8) ◽  
pp. 411
Author(s):  
Maxence Quemener ◽  
Marie Dayras ◽  
Nicolas Frotté ◽  
Stella Debaets ◽  
Christophe Le Meur ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Antimicrobial Activity ◽  
Oceanic Crust ◽  
Gene Clusters ◽  
Multidrug Resistant ◽  
Antimicrobial Activities ◽  
Antimicrobial Compounds ◽  
Human Pathogens ◽  
Biotechnological Potential ◽  
Fungal Isolates

Among the different tools to address the antibiotic resistance crisis, bioprospecting in complex uncharted habitats to detect novel microorganisms putatively producing original antimicrobial compounds can definitely increase the current therapeutic arsenal of antibiotics. Fungi from numerous habitats have been widely screened for their ability to express specific biosynthetic gene clusters (BGCs) involved in the synthesis of antimicrobial compounds. Here, a collection of unique 75 deep oceanic crust fungi was screened to evaluate their biotechnological potential through the prism of their antimicrobial activity using a polyphasic approach. After a first genetic screening to detect specific BGCs, a second step consisted of an antimicrobial screening that tested the most promising isolates against 11 microbial targets. Here, 12 fungal isolates showed at least one antibacterial and/or antifungal activity (static or lytic) against human pathogens. This analysis also revealed that Staphylococcus aureus ATCC 25923 and Enterococcus faecalis CIP A 186 were the most impacted, followed by Pseudomonas aeruginosa ATCC 27853. A specific focus on three fungal isolates allowed us to detect interesting activity of crude extracts against multidrug-resistant Staphylococcus aureus. Finally, complementary mass spectrometry (MS)-based molecular networking analyses were performed to putatively assign the fungal metabolites and raise hypotheses to link them to the observed antimicrobial activities.

scholarly journals A Comparative Study of Antimicrobial Activity of Ethanolic Extract of Tecoma stans and Abutilon indicum Leaves: An In vitro Study

2021 ◽  
pp. 609-615
Author(s):  
M. Binigha ◽  
R. Gayatri Devi ◽  
J. Selavaraj ◽  
A. Jothi Priya
Keyword(s):  
Antimicrobial Activity ◽  
Agar Plate ◽  
Ethanolic Extract ◽  
Flowering Plant ◽  
Diffusion Method ◽  
Bacterial Strains ◽  
Zone Of Inhibition ◽  
The Family ◽  
Abutilon Indicum

Tecoma stans is a flowering plant belonging to the family Bignoniaceae. It is the floral emblem of the Bahamas. Abutilon indicum belongs to the family Malvaceae. It is also called Indian Mallow. Tecoma stans and Abutilon indicumhave antimicrobial activity and can be used in producing antibiotics. There is an urgent need to produce new antibiotics as there is an increase in the development of pathogen resistant drugs. The aim of this study was to compare the antimicrobial activity of ethanolic extract of Tecoma stansand Abutilonindicum leaves. E.faecalis bacteria were maintained in nutrient agar slopes. The powder of the two herbs, were subjected to extraction by Soxhlet extractor and was concentrated to dry residue by Agar well diffusion method. The nutrient broth was inoculated with bacterial strains. The culture was adjusted to 0.5McFarland turbidity standard. Lawn culture was done in Muller-Hinton agar plate and then the plates were dried. A 6 mm diameter well is bored for different concentrations. The extracts were introduced into the well and allowed to stand for 24 hrs. The antibacterial activity was determined by measuring the zone of inhibition. The present finding on the antimicrobial potential of Abutilon indicum and Tecoma stands showed that the two plants have growth inhibitory activity against oral pathogen E. faecalis. Abutilon indicum has a zone of inhibition greater than Tecoma stans. Abutilon indicum exhibited better activity compared to Tecoma stans.

Antimicrobial Activity of Formylchromones: Detection by a Micro-Scale Method

2011 ◽  
Vol 66 (11-12) ◽  
pp. 562-570
Author(s):  
Markku Lehtinen ◽  
Eila Pelttari ◽  
Hannu Elo
Keyword(s):  
Antimicrobial Activity ◽  
High Throughput Screening ◽  
Test Substance ◽  
Minimal Inhibitory Concentrations ◽  
Micro Scale ◽  
Drug Candidates ◽  
Scale Method ◽  
Deep Well ◽  
Quinolone Antibiotics ◽  
Microbial Suspension

We report the antimicrobial activity of formylchromones. These compounds are remote structural analogues of nalidixic acid and quinolone antibiotics, and their activity was investigated by a simple micro-scale method designed for the determination of minimal inhibitory concentrations (MIC) of drug candidates and antibiotics against aerobic bacteria and yeasts. Minimal bactericidal and fungicidal concentrations (MBC and MFC, respectively) were also determined in connection with the MIC determinations. The results obtained were compared with those obtained using classical agar diffusion methodology. In the MIC method, deep-well micro-titration plates are used, covered by silicone sealing mats that allow diffusion of oxygen to the wells. The appropriate broth is pipetted into the wells, followed by a standardized microbial suspension (except for sterile controls) and a dilution series of the test substance or control antibiotic or a mere control solvent. The use of white nontransparent polypropylene plates allows easy visual inspection of microbial growth. For the MBC and MFC methods, samples are taken from all wells that contain a test substance or control antibiotic and do not display growth in the MIC test. The samples are streaked on agar plates, the liquid is allowed to absorb into the agar, and finally the microbes are spread all over the plate with a bent rod. Colony counts are compared with that of the untreated microbial suspension at the beginning of the MIC test. The MIC method is suitable for high-throughput screening

scholarly journals A high‐throughput screening for inhibitors of riboflavin synthase identifies novel antimicrobial compounds to treat brucellosis

FEBS Journal ◽  
2019 ◽  
Vol 286 (13) ◽  
pp. 2522-2535 ◽  
Author(s):  
María Inés Serer ◽  
Mariela del Carmen Carrica ◽  
Jörg Trappe ◽  
Sandra López Romero ◽  
Hernán Ruy Bonomi ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Antimicrobial Compounds

scholarly journals Correlation between the Antimicrobial Activity and Metabolic Profiles of Cell Free Supernatants and Membrane Vesicles Produced by Lactobacillus reuteri DSM 17938

2020 ◽  
Vol 8 (11) ◽  
pp. 1653
Author(s):  
Alessandro Maccelli ◽  
Simone Carradori ◽  
Valentina Puca ◽  
Francesca Sisto ◽  
Paola Lanuti ◽  
...  
Keyword(s):  
Antimicrobial Activity ◽  
Lactobacillus Reuteri ◽  
Membrane Vesicles ◽  
Metabolic Profiles ◽  
Ion Cyclotron Resonance ◽  
Antimicrobial Compounds ◽  
Ionization Source ◽  
Gram Positive ◽  
Gram Negative ◽  
Broth Microdilution Method

The aim of the work is to assess the antimicrobial activities of Cell Free Supernatants (CFS) and Membrane Vesicles (MVs), produced by Lactobacillus reuteri DSM 17938, versus Gram-positive and Gram-negative bacteria and investigate their metabolic profiles. The Minimum Inhibitory Concentration was determined through the broth microdilution method and cell proliferation assay while the Minimum Bactericidal Concentration was determined by Colony Forming Units counts. The characteristics of the antimicrobial compounds were evaluated by pH adjustments, proteinase treatment, and size fractionation of the CFS. The cytotoxicity of CFS was tested on two human cell lines. A detailed snapshot of the L. reuteri metabolism was attained through an untargeted metabolic profiling by means of high resolution Fourier Transform Ion Cyclotron Resonance Mass Spectrometry (FT-ICR MS) coupled with Electrospray Ionization Source (ESI). The results showed (i) a greater efficacy of CFS and its fractions towards Gram-negative compared to Gram-positive bacteria; (ii) an antimicrobial effect related to pH-dependent compounds but not to MVs; (iii) a molecular weight < 3 KDa as well as an a non-proteinaceous nature of the antimicrobial compounds; and (iv) more than 200 and 500 putative metabolites annotated in MVs and supernatants, covering several classes of metabolites, including amino acids, lipids, fatty and organic acids, polyalcohols, nucleotides, and vitamins. Some putative compounds were proposed not only as characteristic of specific fractions, but also possibly involved in antimicrobial activity.

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