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 particulate matter.</li>
		<li>Carry out appropriate serial dilutions of the filtered water for further analysis.</li>
	</ol>
	</li>
</ol>
2. Estimation of the total bacterial load and the antibiotic-resistant bacteria count
<ol>
	<li>Determination of the total bacterial load
	<ol>
		<li>Suspend 18.12 g of R2A Agar, Modified powder in 1,000 mL of double-distilled water, and dissolve the mixture by heating. Autoclave the dissolved mixture at 121 &#176;C, 15 psi for 20 min. Prepare R2A Agar, Modified plates by pouring the appropriate quantity of the autoclaved mixture into sterile Petri plates (e.g., add approximately 20 mL of autoclaved sterile medium to a 90 mm sterile Petri plate).</li>
		<li>Evenly spread 100 &#181;L of the appropriate dilutions of the filtered water sample on the R2A Agar, Modified plate once the medium solidifies. Perform the experiment in duplicate.</li>
		<li>Incubate all the above plates at 35-37 &#176;C for 48 h (vary the temperature and incubation time depending upon the media used for isolation).</li>
		<li>Express the total bacterial load in terms of colony-forming units per milliliter (CFU/mL) using equation (1): 
		<img alt="Equation 1" src="/files/ftp_upload/63934/63934eq01.jpg" style="margin: auto;" />&#160; &#160;(1)</li>
	</ol>
	</li>
	<li>Determination of the AR bacterial count
	<ol>
		<li>Follow steps 2.1.1-2.1.4. However, instead of R2A Agar, Modified plates, use R2A Agar, Modified plates supplemented individually with five different antibiotics, namely cefotaxime (3 &#181;g/mL), ciprofloxacin (0.5 &#181;g/mL), erythromycin (20 &#181;g/mL), kanamycin (15 &#181;g/mL), and vancomycin (3 &#181;g/mL).</li>
		<li>Add the antibiotics separately into tubes containing 20 mL of sterile molten R2A Agar, Modified (with the temperature of the molten R2A Agar, Modified at &#8804;40 &#176;C) to achieve the final antibiotic concentration as mentioned in step 2.2.1.</li>
		<li>Swirl for even mixing and pour onto sterile Petri plates before the agar solidifies. Perform the experiment in duplicate.</li>
		<li>Incubate all the above plates at 35-37 &#176;C for 48 h (if using a different media, the temperature and incubation time may vary).</li>
		<li>For quality control and checking the efficacy of the antibiotics, spread 100 &#181;L of bacterial suspensions of the Escherichia coli ATCC 25922 and Staphylococcus aureus ATCC 29213 strains onto their respective antibiotic-containing R2A Agar, Modified plates (ensure that the density of the fresh culture used for the inoculation is OD = 0.5 at 600 nm).</li>
		<li>Determine the antibiotic-resistant bacterial count in terms of CFU/mL as described in step 2.1.4.</li>
	</ol>
	</li>
	<li>Glycerol stocks of the isolates
	<ol>
		<li>Select morphologically distinct AR colonies.</li>
		<li>Suspend a single isolated colony in 2 mL of sterile Luria-Bertani broth containing the respective antibiotic (e.g., if a colony was selected from a plate containing cefotaxime, inoculate the colony from step 2.3.1 in sterile Luria-Bertani broth containing cefotaxime at its respective concentration).</li>
		<li>Incubate the inoculated tubes at 37 &#176;C at 80 rpm until the OD600 reaches 0.5.</li>
		<li>Prepare glycerol stocks of the isolates by mixing 750 &#181;L of the culture suspension from step 2.3.3 into 250 &#181;L of sterile 100% glycerol under aseptic conditions.</li>
		<li>Store the glycerol stocks at &#8722;80 &#176;C until further analysis. 
		NOTE: For the revival of the cultures from the glycerol stocks, thaw the glycerol stocks at 4 &#176;C. Inoculate a loopful of this stock into 2 mL of sterile Luria-Bertani broth containing the respective antibiotic and allow to grow.</li>
	</ol>
	</li>
</ol>
3. Identification of culturable bacteria by 16S rRNA gene sequencing
<ol>
	<li>Preparation of DNA template from the isolates for PCR 
	NOTE: The protocol described for the preparation of DNA template for PCR for the isolation of crude DNA from the bacteria is given by Carlson et al.10.
	<ol>
		<li>Using a sterile toothpick, take a single, isolated, pure colony of the isolate growing on a Petri plate. Suspend the bacterial colony in 100 &#181;L of sterile double-distilled water in a sterile microcentrifuge tube and boil for 10 min.</li>
		<li>Centrifuge the suspension at 10,000 &#215; g for 2 min to pellet the debris, and transfer the supernatant to a fresh sterile microcentrifuge tube for use as the crude DNA template.</li>
	</ol>
	</li>
	<li>Targeted PCR amplification of the V3 region of the 16S rRNA gene and sequencing
	<ol>
		<li>Prepare 40 &#181;L of the reaction mixture in a PCR tube for PCR amplification, as mentioned in Table 1. 
		NOTE: The DNA preparation should be carried out on an ice block while minimizing the chance of contamination (wear gloves while handling the reagents, and clean the work surface thoroughly with 70% ethanol).</li>
		<li>Place the tube in the thermal block, and run the appropriate program in the PCR thermal cycler. See Table 2 for the standardized PCR cycling conditions and the primer information for the amplification of the V3 regions of the 16S rRNA genes.</li>
		<li>For resolving the amplicons and visualization, carry out agarose gel electrophoresis (AGE). Mix 10 &#181;L of the amplified PCR product and 2 &#181;L of 6x gel loading buffer (Table 3), and load this mixture into wells on 1.5% agarose gel (dissolve 1.5 g of agarose powder in 100 mL of 1x TAE buffer [Table 3]) containing 5 &#181;L of 10 mg/mL ethidium bromide (EtBr) to a final concentration of 0.5 &#181;g/mL EtBr in 100 mL of the agarose gel. 
		CAUTION: EtBr is a potent carcinogen. Gloves should be worn at all times while handling EtBr and gels containing EtBr.</li>
		<li>Add DNA ladder for the estimation of the size of the amplicons.</li>
		<li>Carry out electrophoresis of the gel in a TAE tank buffer at 80-100 V.</li>
		<li>Once the tracking dye runs 3/4 of the gel, stop the electrophoresis, and visualize the amplicon bands under a UV transilluminator.</li>
		<li>Use the PCR product (amplicon) for 16S rRNA gene sequencing to identify the isolate.</li>
		<li>Quantify the amplicon by subjecting it to spectrophotometric analysis using equation (2). 
		<img alt="Equation 2" src="/files/ftp_upload/63934/63934eq02.jpg" style="margin: auto;" />&#160; &#160;(2)</li>
		<li>To check the purity of the DNA, calculate the ratio of A260/A280. 
		NOTE: Ideally, this number should be above 1.5 and, preferably, between 1.8 and 2.0.</li>
		<li>To identify the isolates, compare the sequences that are obtained with sequence databases using an appropriate alignment search tool.</li>
	</ol>
	</li>
</ol>
4. Detection of antibiotic resistance in the isolates using antibiotic susceptibility testing
NOTE: This protocol describes the method for antibiotic susceptibility testing (AST) by disc diffusion. The following antibiotic discs were used: cefotaxime (5 &#181;g), ampicillin (10 &#181;g), levofloxacin (5 &#181;g), chloramphenicol (30 &#181;g), tigecycline (15 &#181;g), ceftriaxone (30 &#181;g), imipenem (10 &#181;g), gentamicin (10 &#181;g), neomycin (10 &#181;g), trimethoprim (5 &#181;g), and ciprofloxacin (5 &#181;g).
<ol>
	<li>Preparation of the inoculum for the AST
	<ol>
		<li>Aseptically inoculate a single, isolated, purified AR colony using a sterile loop in 2 mL of a sterile non-selective medium, such as Luria-Bertani broth (without any antibiotic), and incubate at 37 &#176;C at 80 rpm overnight.</li>
		<li>Resuspend by taking 100-150 &#181;L of the overnight grown culture (approximately, OD600 = 1.8-2.0) in 2 mL of fresh non-selective Luria-Bertani broth medium and incubate for 2-4 h (until the OD600 reaches 0.4-0.5).</li>
		<li>Dilute this freshly grown culture suspension using sterile 0.85% saline solution such that the density of the culture is equal to 0.5 McFarland standard (approximately, OD600 = 0.1), which roughly corresponds to 1-2 &#215; 108 cells/mL.</li>
		<li>Gently mix the bacterial suspension for an even cell distribution.</li>
		<li>Use the above suspension within 15 min of dilution.</li>
	</ol>
	</li>
	<li>Inoculation of the agar plates
	<ol>
		<li>Prepare Mueller-Hinton Agar (MHA) plates for performing the AST by mixing 38 g of MHA in 1,000 mL of double-distilled water, and dissolve the mixture by heating. Autoclave the dissolved mixture at 121 &#176;C, 15 psi for 15 min.</li>
		<li>Ensure that the depth of MHA in the plates is 4 mm (25 mL of medium per plate).</li>
		<li>Simultaneously, remove the antibiotic discs from the freezer and warm them to room temperature. 
		NOTE: The antibiotic discs should be gradually thawed by initially thawing the discs at 4 &#176;C and later at room temperature to reduce any potential danger of condensation on the discs, which may subsequently affect the zone of inhibition (ZOI).</li>
		<li>Under aseptic conditions, dip a sterile cotton swab into the inoculum prepared in step 4.1, and remove excess suspension to avoid over-inoculation of the plates.</li>
		<li>Spread the culture evenly on the plates, starting from the top of the MHA plate and going back and forth from edge to edge. Rotate the plate by 60&#176; while swabbing.</li>
	</ol>
	</li>
	<li>Application of the antibiotic discs
	<ol>
		<li>With the help of flame-sterilized forceps, aseptically transfer the antibiotic discs onto the inoculated MHA plates, and press the discs gently to ensure complete level contact with the agar. 
		NOTE: This procedure has to be done within 15 min of the inoculation of the culture on the plates.</li>
		<li>Place the appropriate number of antibiotic discs on the agar plate by taking into account the organism, the antibiotic used, and the size of the plate to avoid overlapping of the zones of inhibition. 
		NOTE: Four to five discs can be accommodated on a 90 mm circular plate.</li>
	</ol>
	</li>
	<li>Incubation of the plates
	<ol>
		<li>Within 15 min of the application of the antibiotic discs, invert the plates and incubate at 37 &#176;C overnight.</li>
	</ol>
	</li>
	<li>Interpretation of the results
	<ol>
		<li>Measure the ZOI diameter in millimeters (mm), and interpret according to the breakpoint values given by EUCAST11. See the two examples given below.
		<ol>
			<li>The zone diameter breakpoint (mm) for a ciprofloxacin antibiotic disc (5 &#181;g) for Enterobacterales is S &#8805; 25 and R &#60; 22, which means that it is considered to be sensitive (S) if ZOI &#8805; 25 mm, while it is resistant (R) if ZOI &#60; 22 mm. If the ZOI diameter falls between 22 and 25, the isolate is considered to be intermediate (I).</li>
			<li>The zone diameter breakpoint (mm) for a chloramphenicol antibiotic disc (30 &#181;g) for Staphylococcus spp. is S &#8805; 18 and R &#60; 18, which means that it is considered to be sensitive if ZOI &#8805; 18 mm, while it is resistant if ZOI &#60; 18 mm.</li>
		</ol>
		</li>
		<li>Determine the multiple antibiotic resistance (MAR) index by finding the ratio of the number of antibiotics to which the isolate is resistant to the total number of antibiotics to which the isolate is exposed. 
		&#8203;NOTE: For quality control, E. coli ATCC 25922 and S. aureus ATCC 29213 are used as reference strains following the protocol as in step 4.</li>
	</ol>
	</li>
</ol>
5. PCR-based detection of antibiotic resistance genes in the isolates
<ol>
	<li>Use a standard PCR protocol for the identification of ARGs in the isolates. Prepare the DNA template using the protocol given in step 3.1. 
	NOTE: The PCR cycling conditions used in this study were 94 &#176;C for 10 min, followed by 35 cycles of 94 &#176;C for 30 s, annealing for 30 s at the appropriate temperature (as standardized for each primer set), extension at 72 &#176;C for 40 s, and a final extension at 72 &#176;C for 5 min. The reaction mixture is described in Table 4. The list of ARGs, primers, and annealing temperatures is given in Table 5.</li>
	<li>To resolve, visualize, and check the purity of the amplicons, follow steps 3.2.3-3.2.10.</li>
</ol>
6. Whole metagenomic DNA analysis for the identification of the total bacterial diversity and the detection of ARGs in the metagenome
<ol>
	<li>Extraction of the total DNA (metagenome) from the water sample
	<ol>
		<li>Extract the metagenomic DNA from the filtered water samples. 
		NOTE: In the current study, the metagenomic DNA (total DNA) was extracted from the filtered water samples using the referenced DNA isolation kit following the manufacturer&#39;s protocol (see the Table of Materials).</li>
		<li>Check the quality of the DNA by loading 3 &#181;L of the extracted metagenomic DNA onto a 0.8% agarose gel, and run the gel at 80-110 V for approximately 30 min.</li>
		<li>Check for the presence of a single intact band.</li>
		<li>Check the DNA concentration using a fluorometer.</li>
	</ol>
	</li>
	<li>Determination of bacterial diversity and detection of ARGs using whole metagenomic DNA sequencing
	<ol>
		<li>Library preparation and PCR amplification:
		<ol>
			<li>Prepare a paired-end sequencing library using the referenced DNA library prep kit (see the Table of Materials).</li>
			<li>Ready the DNA for adapter ligation by taking 200 ng of DNA and mechanically shearing it into smaller fragments, followed by a continuous step of end-repair in which an &#34;A&#34; is added to the 3&#39; ends.</li>
			<li>Depending on the platform used for sequencing, ligate specific adapters to both ends of the DNA fragments. 
			NOTE: Sequences crucial for binding dual-barcoded libraries to a flow cell for sequencing are present in these adapters. This allows for PCR amplification of the adapter-ligated fragments and binding the standard sequencing primers.</li>
			<li>To check the quality and quantity, analyze the amplified library using a high-sensitivity DNA chip as per the manufacturer&#39;s instructions.</li>
		</ol>
		</li>
		<li>Cluster generation and sequencing:
		<ol>
			<li>Load the amplified library onto the appropriate sequencing platform for cluster generation and subsequent sequencing. 
			NOTE: The library molecules bind to the complementary adapter oligos on the paired-end flow cell. During sequencing, the forward strands are selectively cleaved after the resynthesis of the reverse strand. This copied reverse strand is then sequenced from the opposite end of the fragment.</li>
		</ol>
		</li>
		<li>Bioinformatic analysis:
		<ol>
			<li>Generate scaffolds from the high-quality data using the appropriate platform.</li>
			<li>Subject these scaffolds to bioinformatics analysis for the taxonomic classification and identification of the ARGs. 
			NOTE: The workflow for the whole metagenomic DNA analysis for the identification of the total bacterial diversity and the detection of ARGs in the metagenome is given in Figure 1. A flowsheet of the complete methodology described in the manuscript is given in Figure 2.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Prestinaci, F., Pezzotti, P., Pantosti, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antimicrobial+resistance:+A+global+multifaceted+phenomenon.">Antimicrobial resistance: A global multifaceted phenomenon.</a> Pathogens and Global Health. 109 (7), 309-318 (2015).</li><li>Knight, G., 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=Antimicrobial+resistance+and+COVID-19:+Intersections+and+implications.">Antimicrobial resistance and COVID-19: Intersections and implications.</a> Elife. 10, 64139 (2021).</li><li>Ventola, C. 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. A., Shashidhar, R., Rath, D., Bandekar, J., 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=Characterization+of+multiple+antibiotic+resistance+of+culturable+microorganisms+and+metagenomic+analysis+of+total+microbial+diversity+of+marine+fish+sold+in+retail+shops+in+Mumbai,+India.">Characterization of multiple antibiotic resistance of culturable microorganisms and metagenomic analysis of total microbial diversity of marine fish sold in retail shops in Mumbai, India.</a> Environmental Science and Pollution Research. 25 (7), 6228-6239 (2018).</li><li>Czekalski, N., Gasc&#243;nD&#237;ez, E., B&#252;rgmann, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wastewater+as+a+point+source+of+antibiotic-resistance+genes+in+the+sediment+of+a+freshwater+lake.">Wastewater as a point source of antibiotic-resistance genes in the sediment of a freshwater lake.</a> The ISME Journal. 8 (7), 1381-1390 (2014).</li><li>Kraemer, S., Ramachandran, A., Perron, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibiotic+pollution+in+the+environment:+From+microbial+ecology+to+public+policy.">Antibiotic pollution in the environment: From microbial ecology to public policy.</a> Microorganisms. 7 (6), 180 (2019).</li><li>Edmonds-Wilson, S., Nurinova, N., Zapka, C., Fierer, N., Wilson, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Review+of+human+hand+microbiome+research.">Review of human hand microbiome research.</a> Journal of Dermatological Science. 80 (1), 3-12 (2015).</li><li>de Abreu, V., Perdig&#227;o, J., Almeida, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metagenomic+approaches+to+analyze+antimicrobial+resistance:+An+overview.">Metagenomic approaches to analyze antimicrobial resistance: An overview.</a> Frontiers in Genetics. 11, 575592 (2021).</li><li>Carlson, S., 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=Detection+of+multiresistant+Salmonella+typhimurium+DT104+using+multiplex+and+fluorogenic+PCR.">Detection of multiresistant Salmonella typhimurium DT104 using multiplex and fluorogenic PCR.</a> Molecular and Cellular Probes. 13 (3), 213-222 (1999).</li><li>Breakpoint tables for interpretation of MICs and zone diameters, Version 12.0. European Committee on Antimicrobial Susceptibility Testing Available from: <a target="_blank" href="https://www.eucast.org/fileadmin/src/media/PDFs/EUCAST_files/Breakpoint_tables/v_12.0_Breakpoint_Tables.pdf">https://www.eucast.org/fileadmin/src/media/PDFs/EUCAST_files/Breakpoint_tables/v_12.0_Breakpoint_Tables.pdf</a> (2022)</li><li>Bharti, R., Grimm, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+challenges+and+best-practice+protocols+for+microbiome+analysis.">Current challenges and best-practice protocols for microbiome analysis.</a> Briefings in Bioinformatics. 22 (1), 178-193 (2019).</li><li>Choo, J., Leong, L., Rogers, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sample+storage+conditions+significantly+influence+faecal+microbiome+profiles.">Sample storage conditions significantly influence faecal microbiome profiles.</a> Scientific Reports. 5, 16350 (2015).</li><li>Clinical and Laboratory Standards Institute. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically, 11th edition. , (2015).</li><li>Bayot, M., Bragg, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antimicrobial+Susceptibility+Testing.">Antimicrobial Susceptibility Testing.</a> StatPearls. , (2021).</li><li>Joseph, A. 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><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 &amp;deg;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 &amp;deg;C)&amp;nbsp;</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&amp;nbsp; and carrying out electrophoresis&amp;nbsp;</td></tr><tr><td>Electrophoresis Power pack with electrodes</td><td>Genei</td><td>NA</td><td>For running the AGE&amp;nbsp;</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>&lt;em&gt;Escherichia coli&lt;/em&gt; ATCC 25922&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;</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)&amp;nbsp;</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&amp;nbsp;</td><td>Used for performing PCR for amplification of 16S rRNA region and various Antibiotic Resistance genes</td></tr><tr><td>Primers&amp;nbsp;</td><td>Xcelris</td><td>NA</td><td>For PCR amplication&amp;nbsp;</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&amp;nbsp;</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&amp;nbsp;</td></tr><tr><td>&lt;em&gt;Staphylococcus aureus&lt;/em&gt; 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&amp;nbsp;</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...

Watch this Scientific Journal Video about Isolation and Identification of Waterborne Antibiotic-Resistant Bacteria and Molecular Characterization of their Antibiotic Resistance Genes at JoVE.com

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 ...

isolation-identification-waterborne-antibiotic-resistant-bacteria

Research

JoVE Journal

Environment

6.3K Views.  University of Mumbai. Onze methode beantwoordt belangrijke vragen, zoals wat is de totale belasting van antibioticaresistente bacteriën in het watermonster? Wat is de identiteit van deze bacteriën en wat zijn verschillende soorten antibioticaresistente eigenschappen en genen die ze dragen? Het voordeel van ons protocol is dat het zelfs bacteriën kan detecteren die niet in het laboratorium kunnen worden gekweekt, die een aanzienlijk deel van de totale bacteriën in het watermonster vormen.Vandaar dat theo...

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

Screening Foodstuffs for Class 1 Integrons and Gene Cassettes

Antibiotic resistance is one of the greatest threats to health in the 21st century. Acquisition of resistance genes via lateral gene transfer is a major factor in the spread of diverse resistance mechanisms. Amongst the DNA elements facilitating lateral transfer, the class 1 integrons have largely been responsible for spreading antibiotic resistance determinants amongst Gram negative pathogens. In total, these integrons have acquired and disseminated over 130 different antibiotic resistance genes. With continued antibiotic use, class 1 integrons have become ubiquitous in commensals and pathogens of humans and their domesticated animals. As a consequence, they can now be found in all human waste streams, where they continue to acquire new genes, and have the potential to cycle back into humans via the food chain. This protocol details a streamlined approach for detecting class 1 integrons and their associated resistance gene cassettes in foodstuffs, using culturing and PCR. Using this protocol, researchers should be able to: collect and prepare samples to make enriched cultures and screen for class 1 integrons; isolate single bacterial colonies to identify integron-positive isolates; identify bacterial species that contain class 1 integrons; and characterize these integrons and their associated gene cassettes.

This protocol describes the detection of class 1 integrons and their associated gene cassettes in foodstuffs.

Antibiotic resistance is one of the greatest threats to health in the 21st century. Acquisition of resistance genes via lateral gene transfer is a major ...

One-day Workflow Scheme for Bacterial Pathogen Detection and Antimicrobial Resistance Testing from Blood Cultures

Bloodstream infections are associated with high mortality rates because of the probable manifestation of sepsis, severe sepsis and septic shock1. Therefore, rapid administration of adequate antibiotic therapy is of foremost importance in the treatment of bloodstream infections. The critical element in this process is timing, heavily dependent on the results of bacterial identification and antibiotic susceptibility testing. Both of these parameters are routinely obtained by culture-based testing, which is time-consuming and takes on average 24-48 hours2, 4. The aim of the study was to develop DNA-based assays for rapid identification of bloodstream infections, as well as rapid antimicrobial susceptibility testing. The first assay is a eubacterial 16S rDNA-based real-time PCR assay complemented with species- or genus-specific probes5. Using these probes, Gram-negative bacteria including Pseudomonas spp., Pseudomonas aeruginosa and Escherichia coli as well as Gram-positive bacteria including Staphylococcus spp., Staphylococcus aureus, Enterococcus spp., Streptococcus spp., and Streptococcus pneumoniae could be distinguished. Using this multiprobe assay, a first identification of the causative micro-organism was given after 2 h.
Secondly, we developed a semi-molecular assay for antibiotic susceptibility testing of S. aureus, Enterococcus spp. and (facultative) aerobe Gram-negative rods6. This assay was based on a study in which PCR was used to measure the growth of bacteria7. Bacteria harvested directly from blood cultures are incubated for 6 h with a selection of antibiotics, and following a Sybr Green-based real-time PCR assay determines inhibition of growth. The combination of these two methods could direct the choice of a suitable antibiotic therapy on the same day (Figure 1). In conclusion, molecular analysis of both identification and antibiotic susceptibility offers a faster alternative for pathogen detection and could improve the diagnosis of bloodstream infections.

The design of a straightforward one-day workflow scheme for bacterial pathogen diagnostics enables the rapid recognition of bloodstream infections. The inclusion of eight clinically relevant bacterial targets and their antibiotic resistance profiles offers the clinician an initial insight on the same day, which can lead to more adequate therapy.

Bloodstream infections are associated with high mortality rates because of the probable manifestation of sepsis, severe sepsis and septic shock1. ...

Immunology and Infection

Biosensor for Detection of Antibiotic Resistant Staphylococcus Bacteria

A structurally transformed lytic bacteriophage having a broad host range of Staphylococcus aureus strains and a penicillin-binding protein (PBP 2a) antibody conjugated latex beads have been utilized to create a biosensor designed for discrimination of methicillin resistant (MRSA) and sensitive (MSSA) S. aureus species 1,2. The lytic phages have been converted into phage spheroids by contact with water-chloroform interface. Phage spheroid monolayers have been moved onto a biosensor surface by Langmuir-Blodgett (LB) technique 3. The created biosensors have been examined by a quartz crystal microbalance with dissipation tracking (QCM-D) to evaluate bacteria-phage interactions. Bacteria-spheroid interactions led to reduced resonance frequency and a rise in dissipation energy for both MRSA and MSSA strains. After the bacterial binding, these sensors have been further exposed to the penicillin-binding protein antibody latex beads. Sensors analyzed with MRSA responded to PBP 2a antibody beads; although sensors inspected with MSSA gave no response. This experimental distinction determines an unambiguous discrimination between methicillin resistant and sensitive S. aureus strains. Equally bound and unbound bacteriophages suppress bacterial growth on surfaces and in water suspensions. Once lytic phages are changed into spheroids, they retain their strong lytic activity and show high bacterial capture capability. The phage and phage spheroids can be utilized for testing and sterilization of antibiotic resistant microorganisms. Other applications may include use in bacteriophage therapy and antimicrobial surfaces.

Lytic phage biosensors and antibody beads are able to discriminate between methicillin resistant (MRSA) and sensitive staphylococcus bacteria. The phages were immobilized by a Langmuir-Blodgett method onto a surface of a quartz crystal microbalance sensor and worked as broad range staphylococcus probes. Antibody beads recognize MRSA.

A structurally transformed lytic bacteriophage having a broad host range of Staphylococcus aureus strains and a penicillin-binding protein (PBP 2a) ...

Bioengineering

Isolation, Propagation, and Identification of Bacterial Species with Hydrocarbon Metabolizing Properties from Aquatic Habitats

Hydrocarbon pollutants are recalcitrant to degradation and their accumulation in the environment is toxic to all life forms. Bacteria encode numerous catalytic enzymes and are naturally capable of metabolizing hydrocarbons. Scientists harness biodiversity in aquatic ecosystems to isolate bacteria with biodegradation and bioremediation potential. Such isolates from the environment provide a rich set of metabolic pathways and enzymes, which can be further utilized to scale up the degradation process at an industrial scale. In this article, we outline the general process of isolation, propagation, and identification of bacterial species from aquatic habitats and screen their ability to utilize hydrocarbons as the sole carbon source in vitro using simple techniques. The present protocol describes the isolation of various bacterial species and their subsequent identification using the 16S rRNA analysis. The protocol also presents steps for characterizing the hydrocarbon degrading potential of bacterial isolates. This protocol will be useful for researchers trying to isolate bacterial species from environmental habitats for their biotechnological applications.

We present the process of isolating, propagating, and characterizing hydrocarbon-degrading bacteria from aquatic habitats. The protocol outlines bacterial isolation, identification by the 16S rRNA method, and testing of their hydrocarbon-degrading potential. This article would help researchers in characterizing microbial biodiversity in environmental samples, and specifically screen for microbes with bioremediation potential.

Hydrocarbon pollutants are recalcitrant to degradation and their accumulation in the environment is toxic to all life forms. Bacteria encode numerous ...

Characterization of a Pathogenic Escherichia coli Strain Derived from Oreochromis spp. Farms Using Whole-Genome Sequencing

Aquaculture is one of the fastest-growing food-producing sectors worldwide and tilapia (Oreochromis spp.) farming constitutes the major freshwater fish variety cultured. Because aquaculture practices are susceptible to microbial contamination derived from anthropogenic sources, extensive antibiotic usage is needed, leading to aquaculture systems becoming an important source of antibiotic-resistant and pathogenic bacteria of clinical relevance such as Escherichia coli (E. coli). Here, the antimicrobial resistance, virulence, and mobilome features of a pathogenic E. coli strain, recovered from inland farmed Oreochromis spp., were elucidated through whole-genome sequencing (WGS) and in silico analysis. Antimicrobial susceptibility testing (AST) and WGS were performed. Furthermore, phylogenetic group, serotype, multilocus sequence typing (MLST), acquired antimicrobial resistance, virulence, plasmid, and prophage content were determined using diverse available web tools. The E. coli isolate only exhibited intermediate susceptibility to ampicillin and was characterized as ONT:H21-B1-ST40 strain by WGS-based typing. Although only a single antimicrobial resistance-related gene was detected [mdf(A)], several virulence-associated genes (VAGs) from the atypical enteropathogenic E. coli (aEPEC) pathotype were identified. Additionally, the cargo of plasmid replicons from large plasmid groups and 18 prophage-associated regions were detected. In conclusion, the WGS characterization of an aEPEC isolate, recovered from a fish farm in Sinaloa, Mexico, allows insights into its pathogenic potential and the possible human health risk of consuming raw aquacultural products. It is necessary to exploit next-generation sequencing (NGS) techniques for studying environmental microorganisms and to adopt a one health framework to learn how health issues originate.

Characterization of a Pathogenic Escherichia coli Strain Derived from Oreochromis spp. Farms Using Whole-Genome Sequencing

The feasibility of whole-genome sequencing (WGS) strategies using benchtop instruments has simplified the genome interrogation of every microbe of public health relevance in a lab setting. A methodological adaptation of the workflow for bacterial WGS is described and a bioinformatics pipeline for analysis is also presented.

Aquaculture is one of the fastest-growing food-producing sectors worldwide and tilapia (Oreochromis spp.) farming constitutes the major freshwater fish ...

Genetics

Identification of Rare Bacterial Pathogens by 16S rRNA Gene Sequencing and MALDI-TOF MS

There are a number of rare and, therefore, insufficiently described bacterial pathogens which are reported to cause severe infections especially in immunocompromised patients. In most cases only few data, mostly published as case reports, are available which investigate the role of such pathogens as an infectious agent. Therefore, in order to clarify the pathogenic character of such microorganisms, it is necessary to conduct epidemiologic studies which include large numbers of these bacteria. The methods used in such a surveillance study have to meet the following criteria: the identification of the strains has to be accurate according to the valid nomenclature, they should be easy to handle (robustness), economical in routine diagnostics and they have to generate comparable results among different laboratories. Generally, there are three strategies for identifying bacterial strains in a routine setting: 1) phenotypic identification characterizing the biochemical and metabolic properties of the bacteria, 2) molecular techniques such as 16S rRNA gene sequencing and 3) mass spectrometry as a novel proteome based approach. Since mass spectrometry and molecular approaches are the most promising tools for identifying a large variety of bacterial species, these two methods are described. Advances, limitations and potential problems when using these techniques are discussed.

Matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) and molecular techniques (16S rRNA gene sequencing) permit the identification of rare bacterial pathogens in routine diagnostics. The goal of this protocol lies in the combination of both techniques which leads to more accurate and reliable data.

There are a number of rare and, therefore, insufficiently described bacterial pathogens which are reported to cause severe infections especially in ...

Atomic Force Microscopy Combined with Infrared Spectroscopy as a Tool to Probe Single Bacterium Chemistry

Atomic Force Microscopy-Infrared Spectroscopy (AFM-IR) is a novel combinatory technique, enabling simultaneous characterization of physical properties and chemical composition of sample with nanoscale resolution. By combining AFM with IR, the spatial resolution limitation of conventional IR is overcome, enabling a resolution of 20&#8211;100 nm to be achieved. This opens the door for a broad array of new applications of IR toward probing samples smaller than several micrometers, previously unachievable by means of conventional IR microscopy. AFM-IR is eminently suited for bacterial research, providing both spectral and spatial information at the single cell and intracellular level. The increasing global health concerns and unfavorable future prediction regarding bacterial infections, and especially, rapid development of antimicrobial resistance, has created an urgent need for a research tool capable of phenotypic probing at the single cell and subcellular level. AFM-IR offers the potential to address this need, by enabling detail characterization of chemical composition of a single bacterium. Here, we provide a complete protocol for sample preparation and data acquisition of single spectra and mapping modality, for the application of AFM-IR toward bacterial studies.

Atomic Force Microscopy-Infrared Spectroscopy (AFM-IR) provides a powerful platform for bacterial studies, enabling to achieve nanoscale resolution. Both, mapping of subcellular changes (e.g., upon cell division) as well as comparative studies of chemical composition (e.g., arising from drug resistance) can be conducted at a single cell level in bacteria.

Atomic Force Microscopy-Infrared Spectroscopy (AFM-IR) is a novel combinatory technique, enabling simultaneous characterization of physical properties and ...

Chemistry

Identification of Antibacterial Immunity Proteins in Escherichia coli using MALDI-TOF-TOF-MS/MS and Top-Down Proteomic Analysis

This protocol identifies the immunity proteins of the bactericidal enzymes: colicin E3 and bacteriocin, produced by a pathogenic Escherichia coli strain using antibiotic induction, and identified by MALDI-TOF-TOF tandem mass spectrometry and top-down proteomic analysis with software developed in-house. The immunity protein of colicin E3 (Im3) and the immunity protein of bacteriocin (Im-Bac) were identified from prominent b- and/or y-type fragment ions generated by the polypeptide backbone cleavage (PBC) on the C-terminal side of aspartic acid, glutamic acid, and asparagine residues by the aspartic acid effect fragmentation mechanism. The software rapidly scans in silico protein sequences derived from the whole genome sequencing of the bacterial strain. The software also iteratively removes amino acid residues of a protein sequence in the event that the mature protein sequence is truncated. A single protein sequence possessed mass and fragment ions consistent with those detected for each immunity protein. The candidate sequence was then manually inspected to confirm that all detected fragment ions could be assigned. The N-terminal methionine of Im3 was post-translationally removed, whereas Im-Bac had the complete sequence. In addition, we found that only two or three non-complementary fragment ions formed by PBC are necessary to identify the correct protein sequence. Finally, a promoter (SOS box) was identified upstream of the antibacterial and immunity genes in a plasmid genome of the bacterial strain.

Identification of Antibacterial Immunity Proteins in Escherichia coli using MALDI-TOF-TOF-MS/MS and Top-Down Proteomic Analysis

Here we present a protocol for the rapid identification of proteins produced by genomically sequenced pathogenic bacteria using MALDI-TOF-TOF tandem mass spectrometry and top-down proteomic analysis with software developed in-house. Metastable protein ions fragment because of the aspartic acid effect and this specificity is exploited for protein identification.

This protocol identifies the immunity proteins of the bactericidal enzymes: colicin E3 and bacteriocin, produced by a pathogenic Escherichia coli strain ...

Antibiotic Dereplication Using the Antibiotic Resistance Platform

One of the main challenges in the search for new antibiotics from natural product extracts is the re-discovery of common compounds. To address this challenge, dereplication, which is the process of identifying known compounds, is performed on samples of interest. Methods for dereplication such as analytical separation followed by mass spectrometry are time-consuming and resource-intensive. To improve the dereplication process, we have developed the antibiotic resistance platform (ARP). The ARP is a library of approximately 100 antibiotic resistance genes that have been individually cloned into Escherichia coli. This strain collection has many applications, including a cost-effective and facile method for antibiotic dereplication. The process involves the fermentation of antibiotic-producing microbes on the surface of rectangular Petri dishes containing solid medium, thereby allowing for the secretion and diffusion of secondary metabolites through the medium. After a 6 day fermentation period, the microbial biomass is removed, and a thin agar-overlay is added to the Petri dish to create a smooth surface and enable the growth of the E. coli indicator strains. Our collection of ARP strains is then pinned onto the surface of the antibiotic-containing Petri dish. The plate is next incubated overnight to allow for E. coli growth on the surface of the overlay. Only strains containing resistance to a specific antibiotic (or class) grow on this surface enabling rapid identification of the produced compound. This method has been successfully used for the identification of producers of known antibiotics and as a means to identify those producing novel compounds.

We describe a platform that utilizes a library of isogenic antibiotic resistant Escherichia coli for the dereplication of antibiotics. The identity of an antibiotic produced by bacteria or fungi can be deduced by the growth of E. coli expressing its respective resistance gene. This platform is economically effective and time-efficient.

One of the main challenges in the search for new antibiotics from natural product extracts is the re-discovery of common compounds. To address this ...

Visualization of Bacterial Resistance using Fluorescent Antibiotic Probes

Fluorescent antibiotics are multipurpose research tools that are readily used for the study of antimicrobial resistance, due to their significant advantage over other methods. To prepare these probes, azide derivatives of antibiotics are synthesized, then coupled with alkyne-fluorophores using azide-alkyne dipolar cycloaddition by click chemistry. Following purification, the antibiotic activity of the fluorescent antibiotic is tested by minimum inhibitory concentration assessment. In order to study bacterial accumulation, either spectrophotometry or flow cytometry may be used, allowing for much simpler analysis than methods relying on radioactive antibiotic derivatives. Furthermore, confocal microscopy can be used to examine localization within the bacteria, affording valuable information about mode of action and changes that occur in resistant species. The use of fluorescent antibiotic probes in the study of antimicrobial resistance is a powerful method with much potential for future expansion.

Fluorescently tagged antibiotics are powerful tools that can be used to study multiple aspects of antimicrobial resistance. This article describes the preparation of fluorescently tagged antibiotics and their application to studying antibiotic resistance in bacteria. Probes can be used to study mechanisms of bacterial resistance (e.g., efflux) by spectrophotometry, flow cytometry, and microscopy.

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

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