Name: characterization of a pathogenic escherichia coli strain derived from oreochromis spp. farms using whole-genome sequencing
Uploaded: 2022-12-23T13:40:00
Description: Watch this Scientific Journal Video about Characterization of a Pathogenic Escherichia coli Strain Derived from Oreochromis spp. Farms Using Whole-Genome Sequencing at JoVE.com

To the National Council of Science and Technology of Mexico (CONACyT by its acronym in Spanish) for the Doctoral scholarship awarded to Jos&#233; Antonio Maga&#241;a-Liz&#225;rraga [No. 481143].

NOTE: The E. coli strain ACM5 was recovered by processing and culturing the fish sample for fecal coliform (FC) determination12. During the fish sampling, fish did not show clinical signs of disease, bacterial, or fungal infection, and a mean temperature of 22.3 &#176;C prevailed. After isolation, the E. coli isolate was subjected to biochemical testing and cryopreserved in brain heart infusion (BHI) broth with DMSO (8% v/v) as a cryoprotective agent.
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This study presents an adaptation of the bacterial WGS workflow using a benchtop sequencer and a pipeline for genomic characterization of a pathogenic E. coli variant. Depending on the sequencing platform used, the turnaround times (TATs) for wet laboratory procedures (bacterial culturing, gDNA extraction, library preparation, and sequencing) and sequence analysis could vary, particularly if slow-growing bacteria are studied. Following the protocol for WGS described above, the TAT was within 4 days, which is com...

An erratum was issued for:&#160;<a href="https://www.jove.com/t/64404">Characterization of a Pathogenic Escherichia coli Strain Derived from Oreochromis spp. Farms Using Whole-Genome Sequencing</a>. The Authors section was updated from:
Jos&#233; Antonio Maga&#241;a-Liz&#225;rraga1, 
Bruno G&#243;mez-Gil2, 
Julissa Enciso-Ibarra2, 
Mar&#237;a Elena B&#225;ez-Flores1 
1Unidad de Investigaciones en Salud P&#250;blica &#8220;Dra. Kaethe Willms&#8221;, Facultad de Ciencias Qu&#237;mico Biol&#243;gicas, Universidad Aut&#243;noma de Sinaloa, Ciudad Universitaria 
2Centro de Investigaci&#243;n en Alimentaci&#243;n y Desarrollo, A. C. (CIAD), Unidad Mazatl&#225;n en Acuicultura y Manejo Ambiental
to:
Jos&#233; Antonio Maga&#241;a-Liz&#225;rraga1, 
Bruno G&#243;mez-Gil2, 
Julissa Enciso-Ibarra2, 
Mar&#237;a Elena B&#225;ez-Flores1 
1Unidad de Investigaciones en Salud P&#250;blica &#8220;Dra. Kaethe Willms&#8221;, Facultad de Ciencias Qu&#237;mico Biol&#243;gicas, Universidad Aut&#243;noma de Sinaloa 
2Centro de Investigaci&#243;n en Alimentaci&#243;n y Desarrollo, A. C. (CIAD), Unidad Mazatl&#225;n en Acuicultura y Manejo Ambiental

An erratum was issued for:&#160;<a href="https://www.jove.com/t/64404">Characterization of a Pathogenic Escherichia coli Strain Derived from Oreochromis spp. Farms Using Whole-Genome Sequencing</a>. The Authors section was updated.

Erratum: 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 its production practices are intended to satisfy the rising food demand for human consumption. Global aquaculture production has tripled from 34 million tonnes (Mt) in 1997 to 112 Mt in 20171. The main species groups, contributing to nearly 75% of the production, were seaweed, carps, bivalves, catfish, and tilapia (Oreochromis spp.)1. However, the appearance of diseases caused by microbial entities is unavoidable because of intensive fish farming, leading to potential economic losses2.
Antibiotic usage in fish farming practices is well known for preventing and treating bacterial infections, the main limiting factor in productivity3,4. Nonetheless, residual antibiotics accumulate in aquaculture sediments and water, exerting selective pressure and modifying the fish-associated and the residing bacterial communities5,6,7,8. Consequently, the aquaculture environment serves as a reservoir for antimicrobial resistance genes (ARGs), and the further emergence and spread of antibiotic-resistant bacteria (ARB) in the surrounding milieu9. In addition to the bacterial pathogens commonly observed affecting fish farming practices, members of the Enterobacteriaceae family are often encountered, including human pathogen strains of Enterobacter spp., Escherichia coli, Klebsiella spp., and Salmonella spp.10. E. coli is the most common microorganism isolated from fish meal and water in fish farming11,12,13,14,15.
E. coli is a versatile gram-negative bacterium that inhabits the gastrointestinal tract of mammals and birds as a commensal member of their intestinal microbiota. However, E. coli possess a highly adaptive capacity to colonize and persist in different environmental niches, including soil, sediments, food, and water16. Because of the gene gain and loss through the horizontal gene transfer (HGT) phenomenon, E. coli has rapidly evolved into a well-adapted antibiotic-resistant pathogen, able to cause a broad spectrum of diseases in humans and animals17, 18. Based on the isolation origin, pathogenic variants are defined as intestinal pathogenic E. coli (InPEC) or extra-intestinal pathogenic E. coli (ExPEC). Furthermore, InPEC and ExPEC are subclassified into well-defined pathotypes according to disease manifestation, genetic background, phenotypic traits, and virulence factors (VFs)16,17,19.
Traditional culture and molecular techniques for pathogenic E. coli strains have allowed the rapid detection and identification of different pathotypes. However, they may be time-consuming, laborious, and frequently require high technical training19. Furthermore, no single method can be used to reliably study all pathogenic variants of E. coli because of the complexity of their genetic background. Currently, these drawbacks have been overcome with the advent of high-throughput sequencing (HTS) technologies. Whole-genome sequencing (WGS) approaches and bioinformatic tools have improved the exploration of microbial DNA affordably and at a large scale, facilitating the in-depth characterization of microbes in a single run, including closely related pathogenic variants20,21,22. Depending on the biological questions, several bioinformatics tools, algorithms, and databases can be used to perform data analysis. For instance, if the main goal is to assess the presence of ARGs, VFs, and plasmids, tools such as ResFinder, VirulenceFinder, and PlasmidFinder, along with their associated databases, might be a good starting point. Carri&#231;o et al.22 provided a detailed overview of the different bioinformatics software and related databases applied for microbial WGS analysis, from raw data preprocessing to phylogenetic inference.
Several studies have demonstrated the broad utility of WGS for genome interrogation regarding antimicrobial resistance attributes, pathogenic potential, and tracking of the emergence and evolutionary relationships of clinically relevant variants of E. coli sourced from diverse origins23,24,25,26. WGS has enabled the identification of molecular mechanisms underlying the phenotypic resistance to antimicrobials, including those rare or complex resistance mechanisms. This is through detecting acquired ARG variants, novel mutations in drug-target genes, or promoter regions27,28. Moreover, WGS offers the potential to infer antimicrobial resistance profiles without requiring prior knowledge about the resistance phenotype of a bacterial strain29. Alternatively, WGS has allowed the characterization of the mobile genetic elements (MGEs) carrying both antimicrobial resistance and virulence features, which has driven the bacterial genome evolution of existing pathogens. For instance, the application of WGS during the investigation of the German E. coli outbreak in 2011 resulted in uncovering the unique genomic features of an apparently novel E. coli pathotype; interestingly, those outbreak strains originated from the enteroaggregative E. coli (EAEC) group, which acquired the prophage encoding the Shiga toxin from the enterohemorrhagic E. coli (EHEC) pathotype30.
This work presents a methodological adaptation of the workflow for bacterial WGS using a benchtop sequencer. Moreover, a bioinformatics pipeline is provided using web-based tools to analyze the resulting sequences and further support researchers with limited or no bioinformatics expertise. The described methods allowed elucidation of the antimicrobial resistance, virulence, and mobilome features of a pathogenic E. coli strain ACM5, isolated in 2011 from inland farmed Oreochromis spp. in Sinaloa, Mexico12.

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	<tbody>
		<tr>
			<td>Accublock Mini digital dry bath</td>
			<td>Labnet</td>
			<td>D0100</td>
			<td>Dry bath for incubation of tubes</td>
		</tr>
		<tr>
			<td>Agencourt AMPure XP</td>
			<td>Beckman Coulter</td>
			<td>A63881</td>
			<td>Magnetic beads in solution for DNA library purification</td>
		</tr>
		<tr>
			<td>DeNovix DS-11</td>
			<td>DeNovix Inc.</td>
			<td></td>
			<td>UV-Vis spectophotometer to check the quality of the gDNA extracted</td>
		</tr>
		<tr>
			<td>DNA LoBind Tubes</td>
			<td>Eppendorf</td>
			<td>0030108418</td>
			<td>1.5 mL PCR tubes for DNA library pooling</td>
		</tr>
		<tr>
			<td>DynaMag-2 Magnet</td>
			<td>Invitrogen, Thermo Fisher Scientific</td>
			<td>12321D</td>
			<td>Magnetic microtube rack used during magnetic beads-based DNA purification</td>
		</tr>
		<tr>
			<td>Gram-negative Multibac I.D.</td>
			<td>Diagnostic reseach (Mexico)</td>
			<td>PT-35</td>
			<td>Commercial standard antibiotic disks for antimicrobial susceptibility testing</td>
		</tr>
		<tr>
			<td>MiniSeq Mid Output Kit (300-cycles)</td>
			<td>Illumina</td>
			<td>FC-420-1004</td>
			<td>Reagent cartdrige for paired-end sequencing (2x150)</td>
		</tr>
		<tr>
			<td>MiniSeq System Instrument</td>
			<td>Illumina</td>
			<td>SY-420-1001</td>
			<td>Benchtop sequencer used for Next-generation sequencing</td>
		</tr>
		<tr>
			<td>MiniSpin centrifuge</td>
			<td>Eppendorf</td>
			<td>5452000816</td>
			<td>Standard centrifuge for tubes</td>
		</tr>
		<tr>
			<td>Nextera XT DNA Library Preparation Kit</td>
			<td>Illumina</td>
			<td>FC-131-1024</td>
			<td>Reagents to perform DNA libraries for sequencing. Includes Box 1 and Box 2 reagents for 24 samples</td>
		</tr>
		<tr>
			<td>Nextera XT Index Kit v2</td>
			<td>Illumina</td>
			<td>FC-131-2001, FC-131-2002, FC-131-2003, FC-131-2004</td>
			<td>Index set A, B, C, D</td>
		</tr>
		<tr>
			<td>PhiX Control v3</td>
			<td>Illumina</td>
			<td>FC-110-3001</td>
			<td>DNA library control for sequencing</td>
		</tr>
		<tr>
			<td>Precision waterbath</td>
			<td>LabCare America</td>
			<td>51221081</td>
			<td>Water bath shaker used for bacterial culture</td>
		</tr>
		<tr>
			<td>Qubit 1X dsDNA HS Assay Kit</td>
			<td>Invitrogen, Thermo Fisher Scientific</td>
			<td>Q33231</td>
			<td>Reagents for fluorescence-based DNA quantification assay</td>
		</tr>
		<tr>
			<td>Qubit 2.0 Fluorometer</td>
			<td>Invitrogen, Thermo Fisher Scientific</td>
			<td>Q32866</td>
			<td>Fluorometer used for fluorescence assay&#160;</td>
		</tr>
		<tr>
			<td>Qubit Assay tubes</td>
			<td>Invitrogen, Thermo Fisher Scientific</td>
			<td>Q32856</td>
			<td>0.5 mL PCR tubes for fluorescence-based DNA quantification assay&#160;</td>
		</tr>
		<tr>
			<td>SimpliAmp Thermal Cycler</td>
			<td>Applied Biosystems, Thermo Fisher Scientific</td>
			<td>A24811</td>
			<td>Thermocycler used for DNA library amplification</td>
		</tr>
		<tr>
			<td>Spectronic GENESYS 10 Vis</td>
			<td>Thermo</td>
			<td>335900</td>
			<td>Spectophotometer used for bacterial suspension in antimicrobial susceptibility testing</td>
		</tr>
		<tr>
			<td>ZymoBIOMICS DNA Miniprep Kit</td>
			<td>Zymo Research Inc.</td>
			<td>D4300</td>
			<td>Kit for genomic DNA extraction (50 preps)</td>
		</tr>
	</tbody>
</table>

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.

The antimicrobial susceptibility was determined by the disk diffusion method and interpreted by CLSI breakpoint criteria for 12 antibiotics spanning six distinct antimicrobial classes, that is, aminoglycosides, &#946;-lactams, fluoroquinolones, nitrofurans, phenicols, and folate pathway antagonists. The E. coli ACM5 exhibited sensitivity to all antibiotics except one &#946;-lactam drug. Four &#946;-lactam drugs were tested: ampicillin, carbenicillin, cephalothin, and cefotaxime. Among these, a 14 mm inhibition h...

Watch this Scientific Journal Video about Characterization of a Pathogenic Escherichia coli Strain Derived from Oreochromis spp. Farms Using Whole-Genome Sequencing at JoVE.com

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.

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 ...

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