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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.
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.
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....
1. Sample collection and processing
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.......
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.......
The authors have no conflicting interests to disclose.
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.
....Name | Company | Catalog Number | Comments |
100 bp DNA ladder | Himedia | MBT049-50LN | For estimation of size of the amplicons |
2x PCR Taq mastermix | HiMedia | MBT061-50R | For making PCR reaction mixture |
37 °C Incubator | GS-192, Gayatri Scientific | NA | For incubation of bacteria |
6x Gel Loading Buffer | HiMedia | ML015-1ML | Loading and Tracking dye which helps to weigh down the DNA sample and track the progress of electrophoresis |
Agarose powder | Himedia | MB229-50G | For resolving amplicons during Agarose Gel Electrophoresis (AGE) |
Ampicillin antibiotic disc | HiMedia | SD002 | For performing AST |
Autoclave | Equitron | NA | Required for sterilization of media, glass plates, test tubes, etc |
Bioanalyzer 2100 | Agilent Technologies | NA | To check the quality and quantity of the amplified library |
Bisafety B2 Cabinet | IMSET | IMSET BSC-Class II Type B2 | Used for microbiological work like bacterial culturing, AST etc. |
Cefotaxime antibiotic disc | HiMedia | SD295E-5VL | For performing AST |
Cefotaxime antibiotic powder | HiMedia | TC352-5G | For preparation of antibiotic stock solution required during isolation of antibiotic resistant bacteria |
Ceftriaxone antibiotic disc | HiMedia | SD065 | For performing AST |
Centrifuge Minispin | Eppendorf | Minispin Plus-5453 | Used to pellet the debris during crude DNA preparation |
Chloramphenicol antibiotic disc | HiMedia | SD006-5x50DS | For performing AST |
Ciprofloxacin antibiotic disc | HiMedia | SD060-5x50DS | For performing AST |
Ciprofloxacin antibiotic powder | HiMedia | TC447-5G | For preparation of antibiotic stock solution required during isolation of antibiotic resistant bacteria |
Colorimeter | Quest | NA | For checking the OD of culture suspensions |
Comprehensive Antibiotic Resistance Database (CARD) database | functional annotation of ARGs; https://card.mcmaster.ca/ | ||
Cooling Shaker Incubator | BTL41 Allied Scientific | NA | For incubation of media plates for culturing bacteria |
Deep Freezer (-40 °C) | Haier | DW40L, Haier Biomedicals | For storage of glycerol stocks |
DNA Library Prep Kit | NEB Next Ultra DNA Library Prep Kit for Illumina | NA | Paired-end sequencing library preparation |
EDTA | HiMedia | GRM1195-100G | For preparation of Gel running buffer for Agarose Gel Electrophoresis (AGE) |
Electrophoresis Apparatus | TechResource | 15 cm gel casting tray | For making the agarose gel and carrying out electrophoresis |
Electrophoresis Power pack with electrodes | Genei | NA | For running the AGE |
Erythromycin antibiotic disc | HiMedia | SD222-5VL | For performing AST |
Erythromycin antibiotic powder | HiMedia | CMS528-1G | For preparation of antibiotic stock solution required during isolation of antibiotic resistant bacteria |
Erythromycin antibiotic powder | HiMedia | TC024-5G | For preparation of antibiotic stock solution required during isolation of antibiotic resistant bacteria |
Escherichia coli ATCC 25922 | HiMedia | 0335X-1 | Used as a control while performing AST |
Ethidium Bromide | HiMedia | MB071-1G | Intercalating agent and visualizaion of DNA after electrophoresis under Gel Documentation System |
Fluorometer | Qubit 2.0 | NA | For determining concentration of extracted metagenomic DNA |
Gel Documentation System | BioRad | Used for visualizing PCR amplicons after electrophoresis | |
Gentamicin antibiotic disc | HiMedia | SD170-5x50DS | For performing AST |
Glacial Acetic Acid | HiMedia | AS119-500ML | For preparation of Gel running buffer for Agarose Gel Electrophoresis (AGE) |
Glycerol | HiMedia | GRM1027-500ML | For making glycerol stocks |
Imipenem antibiotic disc | HiMedia | SD073 | For performing AST |
Kaiju Database | NA | NA | For taxonomical classification of reads; https://kaiju.binf.ku.dk/ |
Kanamycin antibiotic disc | HiMedia | SD017-5x50DS | For performing AST |
Kanamycin antibiotic powder | HiMedia | MB105-5G | For preparation of antibiotic stock solution required during isolation of antibiotic resistant bacteria |
Levofloxacin antibiotic disc | HiMedia | SD216-5VL | For performing AST |
Luria Bertani broth | Himedia | M1245-500G | For enrichment of cultures |
McFarland Standards | Himedia | R092-1No | To compare density of culture suspension |
Molecular Biology water | HiMedia | TCL018-500ML | For making PCR reaction mixture |
Mueller-Hinton Agar (MHA) | HiMedia | M173-500G | For performing Antibiotc Susceptibility Testing (AST) |
Neomycin antibiotic disc | HiMedia | SD731-5x50DS | For performing AST |
PCR Gradient Thermal Cycler | Eppendorf | Mastercycler Nexus Gradient 230V/50-60 Hz | Used for performing PCR for amplification of 16S rRNA region and various Antibiotic Resistance genes |
Primers | Xcelris | NA | For PCR amplication |
R2A Agar, Modified | HiMedia | M1743 | For preparation of media plates for isolation of total and antibiotic resistant (AR) bacterial load |
Scaffold generation | CLC Genomics Workbench 6.0 | NA | For generation of scaffolds |
Sequencer | Illumina platform (2 x 150 bp chemistry) | NA | Sequencing of amplified library |
Sodium Chloride | HiMedia | TC046-500G | For preparation of 0.85% saline for serially diluting the water sample |
Soil DNA isolation Kit | Xcelgen | NA | For extraction of whole metagenomic DNA from the filtered water sample |
Staphylococcus aureus subsp. aureus ATCC 29213 | HiMedia | 0365P | Used as a control while performing AST |
Taxonomical Classification | Kaiju ioinformatics tool | NA | For classification of reads into different taxonomic groups from phylum to genus level |
The Comprehensive Antibiotic Resistance Database (CARD) | NA | NA | For functional annotation of ARGs |
Tigecycline antibiotic disc | HiMedia | SD278 | For performing AST |
Trimethoprim antibiotic disc | HiMedia | SD039-5x50DS | For performing AST |
Tris base | HiMedia | TC072-500G | For preparation of Gel running buffer for Agarose Gel Electrophoresis (AGE) |
Vancomycin antibiotic powder | HiMedia | CMS217 | For preparation of antibiotic stock solution required during isolation of antibiotic resistant bacteria |
Weighing Balance | Mettler Toledo | ME204 Mettler Toledo | Used for weighing media powders, reagent powders etc. |
NA - Not Applicable |
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