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

In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Results
  • Discussion
  • Disclosures
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

Here, we present a detailed protocol for the isolation and identification of antibiotic-resistant bacteria from water and the molecular characterization of their antibiotic resistance genes (ARGs). The use of culture-based and non-culture-based (metagenomic analysis) techniques provides complete information about the total bacterial diversity and the total pool of different ARGs present in freshwaters from Mumbai, India.

Abstract

The development and spread of antibiotic resistance (AR) through microbiota associated with freshwater bodies is a major global health concern. In the present study, freshwater samples were collected and analyzed with respect to the total bacterial diversity and AR genes (ARGs) using both conventional culture-based techniques and a high-throughput culture-independent metagenomic approach. This paper presents a systematic protocol for the enumeration of the total and antibiotic-resistant culturable bacteria from freshwater samples and the determination of phenotypic and genotypic resistance in the culturable isolates. Further, we report the use of whole metagenomic analysis of the total metagenomic DNA extracted from the freshwater sample for the identification of the overall bacterial diversity, including non-culturable bacteria, and the identification of the total pool of different ARGs (resistome) in the water body. Following these detailed protocols, we observed a high antibiotic-resistant bacteria load in the range of 9.6 × 105-1.2 × 109 CFU/mL. Most isolates were resistant to the multiple tested antibiotics, including cefotaxime, ampicillin, levofloxacin, chloramphenicol, ceftriaxone, gentamicin, neomycin, trimethoprim, and ciprofloxacin, with multiple antibiotic resistance (MAR) indexes of ≥0.2, indicating high levels of resistance in the isolates. The 16S rRNA sequencing identified potential human pathogens, such as Klebsiella pneumoniae, and opportunistic bacteria, such as Comamonas spp., Micrococcus spp., Arthrobacter spp., and Aeromonas spp. The molecular characterization of the isolates showed the presence of various ARGs, such as blaTEM, blaCTX-M (β-lactams), aadA, aac (6')-Ib (aminoglycosides), and dfr1 (trimethoprims), which was also confirmed by the whole metagenomic DNA analysis. A high prevalence of other ARGs encoding for antibiotic efflux pumps-mtrA, macB, mdtA, acrD, β-lactamases-SMB-1, VIM-20, ccrA, ampC, blaZ, the chloramphenicol acetyltransferase gene catB10, and the rifampicin resistance gene rphB-was also detected in the metagenomic DNA. With the help of the protocols discussed in this study, we confirmed the presence of waterborne MAR bacteria with diverse AR phenotypic and genotypic traits. Thus, whole metagenomic DNA analysis can be used as a complementary technique to conventional culture-based techniques to determine the overall AR status of a water body.

Introduction

Antimicrobial resistance (AMR) has been identified as one of the most pressing global problems. The rapid evolution of AMR and its worldwide spread are one of the greatest threats to human health and the global economy in terms of the health costs associated with it1. The overuse and misuse of antibiotics have led to an increase in AR. This has been highlighted by the COVID-19 pandemic, during which the treatment of associated secondary infections, in many cases, was hugely compromised due to AMR in the affected patients2. Besides the direct use/misuse of antibiotics by humans, the overuse and misuse of antibiotics in ag....

Protocol

1. Sample collection and processing

  1. Sample collection
    1. Collect the appropriate volume of the water sample in sterile sample container(s), ensuring that not more than 3/4 of the container is filled.
    2. Transport the samples to the laboratory under aseptic conditions as soon as possible after collection and immediately process them.
  2. Sample processing
    1. Aseptically filter the water sample through a sterile muslin cloth to remove any particulate matter.
    2. Carry out appropriate serial dilutions of the filtered water for further analysis.

Results

Total bacterial load and antibiotic-resistant (AR) bacteria count
The enumeration of the total bacterial load was carried out by spreading 10−4 to 10−6 fold dilutions of the water samples on R2A Agar, Modified medium. For the enumeration of the AR bacteria count, 10−3 to 10−6 fold dilutions were spread on media plates containing antibiotics (Figure 3). The total and AR bacteria counts were calculated a.......

Discussion

The sample collection and processing play a significant role and might affect the results and interpretation of the study. Hence, to rule out variability in the samples, it is important to carry out sampling at multiple locations of the freshwater body being studied. Maintaining proper aseptic environmental conditions when handling such samples can prevent contamination. Moreover, to prevent changes in the bacterial composition that may influence the quality and quantity of extracted nucleic acids, the transit conditions.......

Disclosures

The authors have no conflicting interests to disclose.

Acknowledgements

This work was partially supported by financial grants from the Department of Science and Technology-Promotion of University Research and Scientific Excellence (DST-PURSE) Scheme of the University of Mumbai. Devika Ghadigaonkar worked as a Project Fellow under the scheme. The technical help provided by Harshali Shinde, Senior Research Fellow under the Department of Science and Technology-Science and Engineering Research Board (DST-SERB) Project no: CRG/2018/003624, is acknowledged.

....

Materials

NameCompanyCatalog NumberComments
100 bp DNA ladderHimediaMBT049-50LNFor estimation of size of the amplicons
2x PCR Taq mastermixHiMediaMBT061-50RFor making PCR reaction mixture
37 °C IncubatorGS-192, Gayatri ScientificNAFor incubation of bacteria
6x Gel Loading BufferHiMediaML015-1MLLoading and Tracking dye which helps to weigh down the DNA sample and track the progress of electrophoresis
Agarose powderHimediaMB229-50GFor resolving amplicons during Agarose Gel Electrophoresis (AGE)
Ampicillin antibiotic discHiMediaSD002For performing AST
AutoclaveEquitronNARequired for sterilization of media, glass plates, test tubes, etc
Bioanalyzer 2100Agilent TechnologiesNATo check the quality and quantity of the amplified library
Bisafety B2 CabinetIMSETIMSET BSC-Class II Type B2Used for microbiological work like bacterial culturing, AST etc.
Cefotaxime antibiotic discHiMediaSD295E-5VLFor performing AST
Cefotaxime antibiotic powderHiMediaTC352-5GFor preparation of antibiotic stock solution required during isolation of antibiotic resistant bacteria
Ceftriaxone antibiotic discHiMediaSD065For performing AST
Centrifuge MinispinEppendorfMinispin Plus-5453Used to pellet the debris during crude DNA preparation
Chloramphenicol antibiotic discHiMediaSD006-5x50DSFor performing AST
Ciprofloxacin antibiotic discHiMediaSD060-5x50DSFor performing AST
Ciprofloxacin antibiotic powderHiMediaTC447-5GFor preparation of antibiotic stock solution required during isolation of antibiotic resistant bacteria
ColorimeterQuestNAFor checking the OD of culture suspensions
Comprehensive Antibiotic Resistance Database (CARD) databasefunctional annotation of ARGs; https://card.mcmaster.ca/
Cooling Shaker IncubatorBTL41 Allied ScientificNAFor incubation of media plates for culturing bacteria
Deep Freezer (-40 °C) HaierDW40L, Haier BiomedicalsFor storage of glycerol stocks
DNA Library Prep KitNEB Next Ultra DNA Library Prep Kit for IlluminaNAPaired-end sequencing library preparation
EDTAHiMediaGRM1195-100GFor preparation of Gel running buffer for Agarose Gel Electrophoresis (AGE)
Electrophoresis ApparatusTechResource15 cm gel casting trayFor making the agarose gel  and carrying out electrophoresis 
Electrophoresis Power pack with electrodesGeneiNAFor running the AGE 
Erythromycin antibiotic discHiMediaSD222-5VLFor performing AST
Erythromycin antibiotic powderHiMediaCMS528-1GFor preparation of antibiotic stock solution required during isolation of antibiotic resistant bacteria
Erythromycin antibiotic powderHiMediaTC024-5GFor preparation of antibiotic stock solution required during isolation of antibiotic resistant bacteria
Escherichia coli ATCC 25922    HiMedia0335X-1Used as a control while performing AST
Ethidium BromideHiMediaMB071-1GIntercalating agent and visualizaion of DNA after electrophoresis under Gel Documentation System
FluorometerQubit 2.0NAFor determining concentration of extracted metagenomic DNA
Gel Documentation SystemBioRadUsed for visualizing PCR amplicons after electrophoresis
Gentamicin antibiotic discHiMediaSD170-5x50DSFor performing AST
Glacial Acetic AcidHiMediaAS119-500MLFor preparation of Gel running buffer for Agarose Gel Electrophoresis (AGE)
GlycerolHiMediaGRM1027-500MLFor making glycerol stocks
Imipenem antibiotic discHiMediaSD073For performing AST
Kaiju DatabaseNANAFor taxonomical classification of reads; https://kaiju.binf.ku.dk/
Kanamycin antibiotic discHiMediaSD017-5x50DSFor performing AST
Kanamycin antibiotic powderHiMediaMB105-5GFor preparation of antibiotic stock solution required during isolation of antibiotic resistant bacteria
Levofloxacin antibiotic discHiMediaSD216-5VLFor performing AST
Luria Bertani brothHimediaM1245-500GFor enrichment of cultures
McFarland StandardsHimediaR092-1NoTo compare density of culture suspension
Molecular Biology waterHiMediaTCL018-500MLFor making PCR reaction mixture
Mueller-Hinton Agar (MHA) HiMediaM173-500GFor performing Antibiotc Susceptibility Testing (AST)
Neomycin antibiotic discHiMediaSD731-5x50DSFor performing AST
PCR Gradient Thermal CyclerEppendorfMastercycler Nexus Gradient 230V/50-60 Hz Used for performing PCR for amplification of 16S rRNA region and various Antibiotic Resistance genes
Primers XcelrisNAFor PCR amplication 
R2A Agar, ModifiedHiMediaM1743For preparation of media plates for isolation of total and antibiotic resistant (AR) bacterial load
Scaffold generationCLC Genomics Workbench 6.0NAFor generation of scaffolds
SequencerIllumina platform (2 x 150 bp chemistry)NASequencing of amplified library
Sodium Chloride HiMediaTC046-500GFor preparation of 0.85% saline for serially diluting the water sample
Soil DNA isolation KitXcelgenNAFor extraction of whole metagenomic DNA from the filtered water sample 
Staphylococcus aureus subsp. aureus ATCC 29213HiMedia0365PUsed as a control while performing AST
Taxonomical ClassificationKaiju ioinformatics toolNAFor classification of reads into different taxonomic groups from phylum to genus level 
The Comprehensive Antibiotic Resistance Database (CARD)NANAFor functional annotation of ARGs
Tigecycline antibiotic discHiMediaSD278For performing AST
Trimethoprim antibiotic discHiMediaSD039-5x50DSFor performing AST
Tris baseHiMediaTC072-500GFor preparation of Gel running buffer for Agarose Gel Electrophoresis (AGE)
Vancomycin antibiotic powderHiMediaCMS217For preparation of antibiotic stock solution required during isolation of antibiotic resistant bacteria
Weighing BalanceMettler ToledoME204 Mettler ToledoUsed for weighing media powders, reagent powders etc.
NA - Not Applicable

References

  1. Prestinaci, F., Pezzotti, P., Pantosti, A. Antimicrobial resistance: A global multifaceted phenomenon. Pathogens and Global Health. 109 (7), 309-318 (2015).
  2. Knight, G., et al. Antimicrobial resistance and COVID-19: Intersect....

Reprints and Permissions

Request permission to reuse the text or figures of this JoVE article

Request Permission

Explore More Articles

Antibiotic resistant BacteriaWater SampleCulture based TechniquesCulture independent TechniquesTotal Bacterial LoadColony Forming UnitsAntibiotic Resistance GenesPCRMolecular Characterization

This article has been published

Video Coming Soon

JoVE Logo

Privacy

Terms of Use

Policies

Research

Education

ABOUT JoVE

Copyright © 2025 MyJoVE Corporation. All rights reserved