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.

1. Sample collection and processing
<ol>
	<li>Sample collection
	<ol>
		<li>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.</li>
		<li>Transport the samples to the laboratory under aseptic conditions as soon as possible after collection and immediately process them.</li>
	</ol>
	</li>
	<li>Sample processing
	<ol>
		<li>Aseptically filter the water sample through a sterile muslin cloth to remove any particulat...

<ol>
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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+antibiotic+resistance+crisis:+Part+1:+Causes+and+threats.">The antibiotic resistance crisis: Part 1: Causes and threats.</a> Pharmacy and Therapeutics. 40 (4), 277-283 (2015).</li><li>Naik, O. A., Shashidhar, R., Rath, D., Bandekar, J. R., Rath, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metagenomic+analysis+of+total+microbial+diversity+and+antibiotic+resistance+of+culturable+microorganisms+in+raw+chicken+meat+and+mung+sprouts+(Phaseolus+aureus)+sold+in+retail+markets+of+Mumbai.">Metagenomic analysis of total microbial diversity and antibiotic resistance of culturable microorganisms in raw chicken meat and mung sprouts (Phaseolus aureus) sold in retail markets of Mumbai.</a> India. Current Science. 113 (1), 71-79 (2017).</li><li>Naik, O. 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A., Odimayo, M. S., Olokoba, L. B., Olokoba, A. B., Popoola, G. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+antibiotic+resistance+index+of+Escherichia+coli+isolates+in+a+tertiary+hospital+in+South-West+Nigeria.">Multiple antibiotic resistance index of Escherichia coli isolates in a tertiary hospital in South-West Nigeria.</a> Medical Journal of Zambia. 44 (4), 225-232 (2017).</li><li>Lorenz, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polymerase+chain+reaction:+Basic+protocol+plus+troubleshooting+and+optimization+strategies.">Polymerase chain reaction: Basic protocol plus troubleshooting and optimization strategies.</a> Journal of Visualized Experiments. (63), e3998 (2012).</li><li>Rolin, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Food+and+human+gut+as+reservoirs+of+transferable+antibiotic+resistance+encoding+genes.">Food and human gut as reservoirs of transferable antibiotic resistance encoding genes.</a> Frontiers in Microbiology. 4, 173 (2013).</li><li>Racewicz, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prevalence+and+characterisation+of+antimicrobial+resistance+genes+and+class+1+and+2+integrons+in+multiresistant+Escherichia+coli+isolated+from+poultry+production.">Prevalence and characterisation of antimicrobial resistance genes and class 1 and 2 integrons in multiresistant Escherichia coli isolated from poultry production.</a> Scientific Reports. 12, 6062 (2022).</li><li>Gebreyes, W., Thakur, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multidrug-resistant+Salmonella+enterica+serovar+Muenchen+from+pigs+and+humans+and+potential+interserovar+transfer+of+antimicrobial+resistance.">Multidrug-resistant Salmonella enterica serovar Muenchen from pigs and humans and potential interserovar transfer of antimicrobial resistance.</a> Antimicrobial Agents and Chemotherapy. 49 (2), 503-511 (2005).</li><li>Li, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prevalence+and+characteristics+of+extended-spectrum+&#946;-lactamase+and+plasmid-mediated+fluoroquinolone+resistance+genes+in+Escherichia+coli+isolated+from+chickens+in+Anhui+Province,+China.">Prevalence and characteristics of extended-spectrum &#946;-lactamase and plasmid-mediated fluoroquinolone resistance genes in Escherichia coli isolated from chickens in Anhui Province, China.</a> PLoS One. 9 (8), 104356 (2014).</li><li>Akers, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aminoglycoside+resistance+and+susceptibility+testing+errors+in+Acinetobacter+baumannii-calcoaceticus+complex.">Aminoglycoside resistance and susceptibility testing errors in Acinetobacter baumannii-calcoaceticus complex.</a> Journal Of Clinical Microbiology. 48 (4), 1132-1138 (2010).</li><li>Ciesielczuk, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Extra-intestinal pathogenic Escherichia coli in the UK: The importance in bacteraemia versus urinary tract infection, colonisation of widespread clones and specific virulence factors. , (2015).</li></ol>

The authors have no conflicting interests to disclose.

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...

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 agriculture and animal husbandry and their inappropriate discharge into the environment, including water bodies, are a major concern3. The rise of new resistance traits and multidrug resistance in bacteria urgently highlights the need for a better understanding of the factors leading to the development of AR and its dissemination. Multiple antibiotic-resistant bacteria, which often carry multiple AR genes (ARGs) on mobile genetic elements such as plasmids, can transfer these resistance genes to non-resistant microorganisms, including potential human pathogens, thus leading to the emergence of superbugs that are untreatable with even last-resort antibiotics4. These multiple antibiotic-resistant bacteria, if present in water ecosystems, can directly enter the human gut via the consumption of contaminated water-based foods such as fish, crabs, and mollusks. Previous studies have shown that the spread of AR bacteria in naturally occurring water systems can also reach other water supplies, including drinking water, and, thus, can enter the human food chain5,6,7.
The aim of the present study is to provide a comprehensive protocol using a combination of culture-based and non-culture-based (whole metagenomic analysis) techniques to obtain complete information about the total bacterial diversity and the total pool of different ARGs present in a water body in Mumbai, India. Conventionally, culture-based techniques have been used to study the bacterial diversity in water bodies. As culturable microorganisms constitute only a small percentage of the total microbiota in any niche, to have a better understanding of the overall status of bacterial diversity and the various resistant traits prevalent in any sample, various culture-based and culture-independent techniques must be used in tandem. One such robust and reliable culture-independent technique is whole metagenomic DNA analysis. This high-throughput method has been successfully utilized in various studies on bacterial diversity or the functional annotations of various ARGs8,9. This technique uses the metagenome (the total genetic material in a sample) as the starting material for various analyses and, hence, is culture-independent. The protocols in the present study can be used for whole metagenomic DNA analysis to obtain information about the total bacterial diversity and various ARGs (resistome) in water samples.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>100 bp DNA ladder</td>
			<td>Himedia</td>
			<td>MBT049-50LN</td>
			<td>For estimation of size of the amplicons</td>
		</tr>
		<tr>
			<td>2x PCR Taq mastermix</td>
			<td>HiMedia</td>
			<td>MBT061-50R</td>
			<td>For making PCR reaction mixture</td>
		</tr>
		<tr>
			<td>37 &#176;C Incubator</td>
			<td>GS-192, Gayatri Scientific</td>
			<td>NA</td>
			<td>For incubation of bacteria</td>
		</tr>
		<tr>
			<td>6x Gel Loading Buffer</td>
			<td>HiMedia</td>
			<td>ML015-1ML</td>
			<td>Loading and Tracking dye which helps to weigh down the DNA sample and track the progress of electrophoresis</td>
		</tr>
		<tr>
			<td>Agarose powder</td>
			<td>Himedia</td>
			<td>MB229-50G</td>
			<td>For resolving amplicons during Agarose Gel Electrophoresis (AGE)</td>
		</tr>
		<tr>
			<td>Ampicillin antibiotic disc</td>
			<td>HiMedia</td>
			<td>SD002</td>
			<td>For performing AST</td>
		</tr>
		<tr>
			<td>Autoclave</td>
			<td>Equitron</td>
			<td>NA</td>
			<td>Required for sterilization of media, glass plates, test tubes, etc</td>
		</tr>
		<tr>
			<td>Bioanalyzer 2100</td>
			<td>Agilent Technologies</td>
			<td>NA</td>
			<td>To check the quality and quantity of the amplified library</td>
		</tr>
		<tr>
			<td>Bisafety B2 Cabinet</td>
			<td>IMSET</td>
			<td>IMSET BSC-Class II Type B2</td>
			<td>Used for microbiological work like bacterial culturing, AST etc.</td>
		</tr>
		<tr>
			<td>Cefotaxime antibiotic disc</td>
			<td>HiMedia</td>
			<td>SD295E-5VL</td>
			<td>For performing AST</td>
		</tr>
		<tr>
			<td>Cefotaxime antibiotic powder</td>
			<td>HiMedia</td>
			<td>TC352-5G</td>
			<td>For preparation of antibiotic stock solution required during isolation of antibiotic resistant bacteria</td>
		</tr>
		<tr>
			<td>Ceftriaxone antibiotic disc</td>
			<td>HiMedia</td>
			<td>SD065</td>
			<td>For performing AST</td>
		</tr>
		<tr>
			<td>Centrifuge Minispin</td>
			<td>Eppendorf</td>
			<td>Minispin Plus-5453</td>
			<td>Used to pellet the debris during crude DNA preparation</td>
		</tr>
		<tr>
			<td>Chloramphenicol antibiotic disc</td>
			<td>HiMedia</td>
			<td>SD006-5x50DS</td>
			<td>For performing AST</td>
		</tr>
		<tr>
			<td>Ciprofloxacin antibiotic disc</td>
			<td>HiMedia</td>
			<td>SD060-5x50DS</td>
			<td>For performing AST</td>
		</tr>
		<tr>
			<td>Ciprofloxacin antibiotic powder</td>
			<td>HiMedia</td>
			<td>TC447-5G</td>
			<td>For preparation of antibiotic stock solution required during isolation of antibiotic resistant bacteria</td>
		</tr>
		<tr>
			<td>Colorimeter</td>
			<td>Quest</td>
			<td>NA</td>
			<td>For checking the OD of culture suspensions</td>
		</tr>
		<tr>
			<td>Comprehensive Antibiotic Resistance Database (CARD) database</td>
			<td></td>
			<td></td>
			<td>functional annotation of ARGs; https://card.mcmaster.ca/</td>
		</tr>
		<tr>
			<td>Cooling Shaker Incubator</td>
			<td>BTL41 Allied Scientific</td>
			<td>NA</td>
			<td>For incubation of media plates for culturing bacteria</td>
		</tr>
		<tr>
			<td>Deep Freezer (-40 &#176;C)&#160;</td>
			<td>Haier</td>
			<td>DW40L, Haier Biomedicals</td>
			<td>For storage of glycerol stocks</td>
		</tr>
		<tr>
			<td>DNA Library Prep Kit</td>
			<td>NEB Next Ultra DNA Library Prep Kit for Illumina</td>
			<td>NA</td>
			<td>Paired-end sequencing library preparation</td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>HiMedia</td>
			<td>GRM1195-100G</td>
			<td>For preparation of Gel running buffer for Agarose Gel Electrophoresis (AGE)</td>
		</tr>
		<tr>
			<td>Electrophoresis Apparatus</td>
			<td>TechResource</td>
			<td>15 cm gel casting tray</td>
			<td>For making the agarose gel&#160; and carrying out electrophoresis&#160;</td>
		</tr>
		<tr>
			<td>Electrophoresis Power pack with electrodes</td>
			<td>Genei</td>
			<td>NA</td>
			<td>For running the AGE&#160;</td>
		</tr>
		<tr>
			<td>Erythromycin antibiotic disc</td>
			<td>HiMedia</td>
			<td>SD222-5VL</td>
			<td>For performing AST</td>
		</tr>
		<tr>
			<td>Erythromycin antibiotic powder</td>
			<td>HiMedia</td>
			<td>CMS528-1G</td>
			<td>For preparation of antibiotic stock solution required during isolation of antibiotic resistant bacteria</td>
		</tr>
		<tr>
			<td>Erythromycin antibiotic powder</td>
			<td>HiMedia</td>
			<td>TC024-5G</td>
			<td>For preparation of antibiotic stock solution required during isolation of antibiotic resistant bacteria</td>
		</tr>
		<tr>
			<td>Escherichia coli ATCC 25922&#160;&#160;&#160;&#160;</td>
			<td>HiMedia</td>
			<td>0335X-1</td>
			<td>Used as a control while performing AST</td>
		</tr>
		<tr>
			<td>Ethidium Bromide</td>
			<td>HiMedia</td>
			<td>MB071-1G</td>
			<td>Intercalating agent and visualizaion of DNA after electrophoresis under Gel Documentation System</td>
		</tr>
		<tr>
			<td>Fluorometer</td>
			<td>Qubit 2.0</td>
			<td>NA</td>
			<td>For determining concentration of extracted metagenomic DNA</td>
		</tr>
		<tr>
			<td>Gel Documentation System</td>
			<td>BioRad</td>
			<td></td>
			<td>Used for visualizing PCR amplicons after electrophoresis</td>
		</tr>
		<tr>
			<td>Gentamicin antibiotic disc</td>
			<td>HiMedia</td>
			<td>SD170-5x50DS</td>
			<td>For performing AST</td>
		</tr>
		<tr>
			<td>Glacial Acetic Acid</td>
			<td>HiMedia</td>
			<td>AS119-500ML</td>
			<td>For preparation of Gel running buffer for Agarose Gel Electrophoresis (AGE)</td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>HiMedia</td>
			<td>GRM1027-500ML</td>
			<td>For making glycerol stocks</td>
		</tr>
		<tr>
			<td>Imipenem antibiotic disc</td>
			<td>HiMedia</td>
			<td>SD073</td>
			<td>For performing AST</td>
		</tr>
		<tr>
			<td>Kaiju Database</td>
			<td>NA</td>
			<td>NA</td>
			<td>For taxonomical classification of reads; https://kaiju.binf.ku.dk/</td>
		</tr>
		<tr>
			<td>Kanamycin antibiotic disc</td>
			<td>HiMedia</td>
			<td>SD017-5x50DS</td>
			<td>For performing AST</td>
		</tr>
		<tr>
			<td>Kanamycin antibiotic powder</td>
			<td>HiMedia</td>
			<td>MB105-5G</td>
			<td>For preparation of antibiotic stock solution required during isolation of antibiotic resistant bacteria</td>
		</tr>
		<tr>
			<td>Levofloxacin antibiotic disc</td>
			<td>HiMedia</td>
			<td>SD216-5VL</td>
			<td>For performing AST</td>
		</tr>
		<tr>
			<td>Luria Bertani broth</td>
			<td>Himedia</td>
			<td>M1245-500G</td>
			<td>For enrichment of cultures</td>
		</tr>
		<tr>
			<td>McFarland Standards</td>
			<td>Himedia</td>
			<td>R092-1No</td>
			<td>To compare density of culture suspension</td>
		</tr>
		<tr>
			<td>Molecular Biology water</td>
			<td>HiMedia</td>
			<td>TCL018-500ML</td>
			<td>For making PCR reaction mixture</td>
		</tr>
		<tr>
			<td>Mueller-Hinton Agar (MHA)&#160;</td>
			<td>HiMedia</td>
			<td>M173-500G</td>
			<td>For performing Antibiotc Susceptibility Testing (AST)</td>
		</tr>
		<tr>
			<td>Neomycin antibiotic disc</td>
			<td>HiMedia</td>
			<td>SD731-5x50DS</td>
			<td>For performing AST</td>
		</tr>
		<tr>
			<td>PCR Gradient Thermal Cycler</td>
			<td>Eppendorf</td>
			<td>Mastercycler Nexus Gradient 230V/50-60 Hz&#160;</td>
			<td>Used for performing PCR for amplification of 16S rRNA region and various Antibiotic Resistance genes</td>
		</tr>
		<tr>
			<td>Primers&#160;</td>
			<td>Xcelris</td>
			<td>NA</td>
			<td>For PCR amplication&#160;</td>
		</tr>
		<tr>
			<td>R2A Agar, Modified</td>
			<td>HiMedia</td>
			<td>M1743</td>
			<td>For preparation of media plates for isolation of total and antibiotic resistant (AR) bacterial load</td>
		</tr>
		<tr>
			<td>Scaffold generation</td>
			<td>CLC Genomics Workbench 6.0</td>
			<td>NA</td>
			<td>For generation of scaffolds</td>
		</tr>
		<tr>
			<td>Sequencer</td>
			<td>Illumina platform (2 x 150 bp chemistry)</td>
			<td>NA</td>
			<td>Sequencing of amplified library</td>
		</tr>
		<tr>
			<td>Sodium Chloride&#160;</td>
			<td>HiMedia</td>
			<td>TC046-500G</td>
			<td>For preparation of 0.85% saline for serially diluting the water sample</td>
		</tr>
		<tr>
			<td>Soil DNA isolation Kit</td>
			<td>Xcelgen</td>
			<td>NA</td>
			<td>For extraction of whole metagenomic DNA from the filtered water sample&#160;</td>
		</tr>
		<tr>
			<td>Staphylococcus aureus subsp. aureus ATCC 29213</td>
			<td>HiMedia</td>
			<td>0365P</td>
			<td>Used as a control while performing AST</td>
		</tr>
		<tr>
			<td>Taxonomical Classification</td>
			<td>Kaiju ioinformatics tool</td>
			<td>NA</td>
			<td>For classification of reads into different taxonomic groups from phylum to genus level&#160;</td>
		</tr>
		<tr>
			<td>The Comprehensive Antibiotic Resistance Database (CARD)</td>
			<td>NA</td>
			<td>NA</td>
			<td>For functional annotation of ARGs</td>
		</tr>
		<tr>
			<td>Tigecycline antibiotic disc</td>
			<td>HiMedia</td>
			<td>SD278</td>
			<td>For performing AST</td>
		</tr>
		<tr>
			<td>Trimethoprim antibiotic disc</td>
			<td>HiMedia</td>
			<td>SD039-5x50DS</td>
			<td>For performing AST</td>
		</tr>
		<tr>
			<td>Tris base</td>
			<td>HiMedia</td>
			<td>TC072-500G</td>
			<td>For preparation of Gel running buffer for Agarose Gel Electrophoresis (AGE)</td>
		</tr>
		<tr>
			<td>Vancomycin antibiotic powder</td>
			<td>HiMedia</td>
			<td>CMS217</td>
			<td>For preparation of antibiotic stock solution required during isolation of antibiotic resistant bacteria</td>
		</tr>
		<tr>
			<td>Weighing Balance</td>
			<td>Mettler Toledo</td>
			<td>ME204 Mettler Toledo</td>
			<td>Used for weighing media powders, reagent powders etc.</td>
		</tr>
		<tr>
			<td>NA - Not Applicable</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation and identification of waterborne antibiotic-resistant bacteria and molecular characterization of their antibiotic resistance genes

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 &#215; 105-1.2 &#215; 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 &#8805;0.2, indicating high levels of resistance in the isolates. The 16S rRNA sequencing identified potential human pathogens, such as Klebsiella pneumoniae,&#160;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 (&#946;-lactams), aadA, aac (6&#39;)-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, &#946;-lactamases-SMB-1, VIM-20, ccrA, ampC, blaZ, the&#160;chloramphenicol acetyltransferase gene catB10, and&#160;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.

Total bacterial load and antibiotic-resistant (AR) bacteria count 
The enumeration of the total bacterial load was carried out by spreading 10&#8722;4 to 10&#8722;6 fold dilutions of the water samples on R2A Agar, Modified medium. For the enumeration of the AR bacteria count, 10&#8722;3 to 10&#8722;6 fold dilutions were spread on media plates containing antibiotics (Figure 3). The total and AR bacteria counts were calculated a...

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Isolation and Identification of Waterborne Antibiotic-Resistant Bacteria and Molecular Characterization of their Antibiotic Resistance Genes

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 ...

Our method answers key questions, such as what is the overall load of antibiotic resistant bacteria in the water sample? What is the identity of these bacteria and what are different types of antibiotic resistant traits and genes they are carrying? The advantage of our protocol is that it can detect even bacteria that cannot be grown in the lab, which constitute a significant proportion of the total bacteria in the water sample.Hence, theoretically, no bacterium or its resistant traits are left out. This combinatorial technique involving both culture-based and culture-independent techniques can be replicated and customized for any sample obtained from sewage, pharmaceutical or hospital effluence, clinical and nonclinical settings, and more. Along with Devika, demonstrating the experimental procedures will be Harshali Shinde and Maitri Mishra, both senior research fellows from my laboratory.To begin, process the water sample aseptically by filtering it through a sterile muslin cloth to remove any particulate matter. Then, serially dilute the filtered water sample for further analysis. To determine the total bacterial load, place 100 microliters of the appropriate dilutions of the filtered water sample on the solidified R2A agar plates and duplicates, and spread evenly.Incubate all the sample spread plates at 35 to 37 degrees Celsius for 48 hours. After the incubation period, count the colonies and express the total bacterial load in colony forming units per milliliter. To determine the antibiotic resistant bacterial count, add the antibiotics separately into tubes containing 20 milliliters of sterile molten R2A agar modified approximately at 40 degrees Celsius.Swirl to mix evenly and pour onto sterile Petri plates before the agar solidifies. For quality control and to check the antibiotics'efficacy, spread 100 microliters of bacterial suspension onto their respective antibiotic containing R2A agar modified plates. Incubate the plates at 35 to 37 degrees Celsius for 48 hours, and then determine the bacterial count.To prepare the DNA template from the isolates for PCR, using a sterile toothpick, take a single isolated pure colony of the isolate growing on a Petri plate. Suspend the bacterial colony in 100 microliters of sterile double distilled water in a micro centrifuge tube and boil it in a boiling water bath or a dry bath for 10 minutes at 100 degrees Celsius. Centrifuge the suspension at 10, 000 g for two minutes to pellet the debris and transfer the supernatant to a fresh sterile microcentrifuge tube to use as the crude DNA template.To amplify the V3 region of the 16S rRNA gene by PCR, prepare 40 microliters of the reaction mixture in a PCR tube. Place the tube in the thermal block, and run the appropriate program in the PCR thermal cycler. For resolving and visualizing the PCR amplicons, mix 10 microliters of the amplified PCR product in two microliters of 6x gel loading buffer, and load this mixture into the wells of a 1.5%agarose gel along with a 100 base paired DNA ladder for estimating the size of the amplicons.Carry out electrophoresis of the gel in the TAE tank buffer at 80 to 100 volts until the tracking dye runs 3/4 of the gel. Then visualize the amplicon bands under a UV transilluminator. To prepare the inoculum, aseptically inoculate a single isolated purified antibiotic resistance colony using a sterile loop in two milliliters of Luria-Bertani, or LB, broth without any antibiotic and incubate at 37 degrees Celsius at 80 rpm overnight.The following day, add 100 to 150 microliters of the overnight grown culture to two milliliters of fresh non-selective LB broth and incubate for two to four hours until the optical density at 600 nanometers reaches 0.4 to 0.5. Dilute the freshly grown culture suspension using sterile 0.85%saline solution and mix gently to distribute the cells evenly to reach a culture density equivalent to 0.5 McFarland standard. Within 15 minutes of dilution, dip a sterile cotton swab into the inoculum and remove the excess suspension to avoid over inoculation of the plates.Then spread the culture evenly on the prepared Mueller Hinton agar plate, starting from the top and going back and forth from edge to edge. Then rotate the plate by 60 degrees, and start swabbing again. To place the antibiotic discs, use a flame sterilized forceps and aseptically transfer the antibiotic discs onto the inoculated agar plates.Press the discs gently to ensure complete level of contact with the agar. Place the appropriate number of antibiotic discs on the agar plate by considering the organism the antibiotic used and the plate size to avoid overlapping the inhibition zones. Within 15 minutes of the antibiotic disc application, invert the plates and incubate them at 37 degrees Celsius overnight.The following day, measure the zone of inhibition diameter in millimeters and interpret it according to the breakpoint values given by EUCAST. The total bacterial load in the water sample was found to be 3.0 x 10 to the ninth CFU/mL, and the antibiotic resistant bacterial load was found to be high. The antibiotic resistance cultural isolates sequenced belonged to the Enterobacteriaceae family with most being Escherichia coli and Klebsiella pneumoniae.In addition, rare opportunistic bacteria were also identified. The antibiotic susceptibility testing by disc diffusion method showed a Multiple Antibiotic Resistance index of approximately 0.2 for eight isolates, indicating a high degree of resistance. However, Comamonas and Arthrobacter species did not show resistance to any of the tested antibiotics.The culturable isolates revealed the presence of antibiotic resistance genes encoding the factors against beta-lactams, trimethoprim, and aminoglycosides. Metagenomic DNA analysis for total bacterial diversity led to the identification of 50 phyla, indicating the high bacterial diversity where Proteobacteria was the most dominant phylum, consisting of the Alphaproteobacteria, Betaproteobacteria, and Gammaproteobacteria classes. At the order level, Burkholderiales was the most prevalent, belonging to the Betaproteobacteria class.At the general level, Pseudomonas, Acinetobacter, Pedobacter, Prosthecobacter, Limnohabitans, Flavobacterium, and Comamonas were some of the abundant bacteria. The metagenomic DNA analysis also revealed more antibiotic resistance genes. In this method, the serial dilution step is critical as any error can lead to a wrong interpretation of the total CFU and the antibiotic resistant bacterial load.Also, the diameter of the zone of inhibition should be measured carefully to correctly interpret if the isolate is resistant or sensitive. Using these protocols, besides water sample, any other sample, be it environmental, food, or clinical, can be studied. Moreover, besides bacteria, other microbial populations in any ecosystem can also be studied using similar approaches.

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5.6K Views.  University of Mumbai. Our method answers key questions, such as what is the overall load of antibiotic resistant bacteria in the water sample? What is the identity of these bacteria and what are different types of antibiotic resistant traits and genes they are carrying? The advantage of our protocol is that it can detect even bacteria that cannot be grown in the lab, which constitute a significant proportion of the total bacteria in the water sample.Hence, theoretically, no bacterium or its resistant traits...