Enhanced Extraction of Low-Molecular Weight DNA from Wastewater for Comprehensive Assessment of Antimicrobial Resistance

Jyotsna Karan; Sreya Mandal; Gulafsha Khan; Harshit Arya; Laasya Samhita

doi:10.3791/66899

Estrazione avanzata di DNA a basso peso molecolare dalle acque reflue per una valutazione complet...

JoVE Journal
Biology

A subscription to JoVE is required to view this content. Sign in or start your free trial.

JoVE Journal Biology

Enhanced Extraction of Low-Molecular Weight DNA from Wastewater for Comprehensive Assessment of Antimicrobial Resistance

Estrazione avanzata di DNA a basso peso molecolare dalle acque reflue per una valutazione completa della resistenza agli antimicrobici

Full Text

1,718 Views

06:54 min

July 19, 2024

DOI: 10.3791/66899-v

Jyotsna Karan¹, Sreya Mandal¹, Gulafsha Khan¹, Harshit Arya¹, Laasya Samhita¹

¹Ashoka University

Please note that some of the translations on this page are AI generated. Click here for the English version.

Overview

This study develops a novel technique for assessing environmental antimicrobial resistance (AMR) by enriching low-molecular-weight extracellular DNA from wastewater samples. The protocol allows for the detection of genomic and horizontally transferred AMR genes, offering a cost-effective, kit-free method for environmental AMR tracking.

Key Study Components

Research Area

Antimicrobial resistance (AMR)
Environmental microbiology
Molecular genetics

Background

The evolution and transmission of antibiotic resistance in environmental settings
Current deficiencies in high yield extraction techniques of low molecular weight DNA
The importance of tracking AMR for public health

Methods Used

PCR and shotgun sequencing for microbial diversity profiling
Use of polyethylene glycol and sodium chloride for DNA extraction
Advanced AMR detection via metabar coding and gene-based panels

Main Results

Enhanced extraction of linear and low molecular weight DNA
Successful capture of both genomic and free DNA fractions associated with AMR
Foundational work for future developments in environmental AMR detection techniques

Conclusions

The study provides an efficient method for assessing environmental AMR
Promotes further research into both genetic and non-genetic factors contributing to antibiotic resistance

Frequently Asked Questions

What is the significance of low-molecular-weight extracellular DNA in AMR?

Low-molecular-weight extracellular DNA serves as a reservoir for AMR genes, thus its enrichment allows better detection and understanding of AMR dynamics.

How does this protocol enhance DNA extraction?

The protocol utilizes polyethylene glycol and sodium chloride to increase the yield of low molecular weight DNA during extraction from wastewater samples.

What are the applications of this research?

This research can help in monitoring trends in AMR, improving public health responses, and developing environmental policies.

What genetic mechanisms are being explored in relation to AMR?

The study compares contributions of horizontal gene transfer and genomic mutations to the development of antibiotic resistance.

Can this technique be developed for commercial use?

Yes, the protocol is designed to be cost-effective and potentially kit-free with further optimization.

How will the results impact future biological research?

This method opens avenues for more comprehensive studies on AMR in various environments, bridging gaps in genetic and environmental research.

What are the future directions for the authors' research?

Future studies will focus on expanding the database for tracking spatial-temporal variations in AMR and exploring non-genetic resistance mechanisms.

Qui, presentiamo una semplice tecnica per valutare la resistenza antimicrobica ambientale (AMR) aumentando la proporzione di DNA extracellulare a basso peso molecolare. Il trattamento precedente con PEG al 20%-30% e NaCl 1,2 M consente di rilevare sia i geni genomici che quelli AMR trasferiti orizzontalmente. Il protocollo si presta a un processo senza kit con un'ulteriore ottimizzazione.

Siamo interessati all'evoluzione, alla trasmissione e ai meccanismi molecolari alla base della resistenza agli antibiotici. In particolare, il lavoro che alimenta questo documento deriva dal nostro interesse per la resistenza ambientale. Attualmente, stiamo cercando di costruire un database locale per tenere traccia della variazione temporale spaziale della resistenza antimicrobica utilizzando i dati di un anno.

Per rilevare e monitorare la resistenza antimicrobica viene utilizzata una combinazione di tecniche basate sulla coltura e sulla genomica. Il DNA dei campioni viene sottoposto a PCR o sequenziamento shotgun per profilare la diversità microbica e rilevare i geni di resistenza. Inoltre, per il rilevamento avanzato della resistenza antimicrobica vengono utilizzati il metabarcoding e i pannelli AMR basati sui geni.

View the full transcript and gain access to thousands of scientific videos