Here, we report the draft genome sequence of three glutaraldehyde-resistant isolates from produced water from hydraulic fracturing operations. The three strains were identified as sp. strain G11, sp. strain G15, and sp. strain G16. The genome sequences of these isolates will provide insights into biocide resistance in hydraulic fracturing operations

Aluko, Dotun

Andersen, Justin

Baldwin, Andrew L

Becker, Cole

Chandler, Grace

Forgrave, Steve

Jones, Madelyn

Klasner, Ina

Martini, Jared

Mason, Noah

Parsons, Ryleigh

Peterson, Nick

Reynolds, Erik

Schroeder, Lydia

Techtmann, Stephen

English

PubMed

Michigan Technological University

Michigan Technological University Digital Commons @ Michigan Tech Michigan Tech Publications 2-17-2022 Draft Genome Sequencing of Three Glutaraldehyde-Tolerant Bacteria from Produced Water from Hydraulic Fracturing Stephen Techtmann Michigan Technological University, smtechtm@mtu.edu Andrew L. Baldwin Michigan Technological University Dotun Aluko Michigan Technological University, ajaluko@mtu.edu Justin Andersen Michigan Technological University Cole Becker Michigan Technological University, coleb@mtu.edu See next page for additional authors Follow this and additional works at: https://digitalcommons.mtu.edu/michigantech-p  Part of the Biology Commons Recommended Citation Techtmann, S., Baldwin, A. L., Aluko, D., Andersen, J., Becker, C., Chandler, G., Forgrave, S., Jones, M., Klasner, I., Martini, J., Mason, N., Parsons, R., Peterson, N., Reynolds, E., & Schroeder, L. (2022). Draft Genome Sequencing of Three Glutaraldehyde-Tolerant Bacteria from Produced Water from Hydraulic Fracturing. Microbiology resource announcements, 11(2), e0123221. http://doi.org/10.1128/mra.01232-21 Retrieved from: https://digitalcommons.mtu.edu/michigantech-p/15696 Follow this and additional works at: https://digitalcommons.mtu.edu/michigantech-p  Part of the Biology Commons Authors Stephen Techtmann, Andrew L. Baldwin, Dotun Aluko, Justin Andersen, Cole Becker, Grace Chandler, Steve Forgrave, Madelyn Jones, Ina Klasner, Jared Martini, Noah Mason, Ryleigh Parsons, Nick Peterson, Erik Reynolds, and Lydia Schroeder This article is available at Digital Commons @ Michigan Tech: https://digitalcommons.mtu.edu/michigantech-p/15696 Draft Genome Sequencing of Three Glutaraldehyde-TolerantBacteria from Produced Water from Hydraulic FracturingStephen M. Techtmann,a Andrew L. Baldwin,a Dotun Aluko,a Justin Andersen,a Cole Becker,a Grace Chandler,a Steve Forgrave,aMadelyn Jones,a Ina Klasner,a Jared Martini,a Noah Mason,a Ryleigh Parsons,a Nick Peterson,a Erik Reynolds,a Lydia SchroederaaDepartment of Biological Sciences, Michigan Technological University, Houghton, Michigan, USAABSTRACT Here, we report the draft genome sequence of three glutaraldehyde-re-sistant isolates from produced water from hydraulic fracturing operations. The threestrains were identified as Marinobacter sp. strain G11, Halomonas sp. strain G15, andBacillus sp. strain G16. The genome sequences of these isolates will provide insightsinto biocide resistance in hydraulic fracturing operations.These three strains were isolated from produced water collected from hydraulic fracturingoperations in the Permian basin of West Texas, USA. Hydraulic fracturing—also known asunconventional oil and gas production (UOG)—is the process of injecting water, sand, andother chemicals into oil -and gas-harboring shale formations (1). This process allows for recov-ery of oil and gas from previously difficult to access hydrocarbon basins. One of the chemicaltypes commonly used in UOG operations is biocides (2). Biocides are antimicrobial chemicalsused to control microbial growth in diverse settings. Glutaraldehyde is one of the most com-monly used biocides in UOG operations (1, 2). There is concern about the potential for biocideuse in UOG operations to lead to development of antimicrobial-resistant bacteria in the forma-tion as well as in adjacent streams (3).To better understand the diversity of biocide-resistant bacteria in the produced water, weisolated glutaraldehyde-tolerant strains of bacteria. Water samples were treated with 100 ppmof glutaraldehyde and incubated for 1 h. The samples were plated on nutrient agar and incu-bated at 30°C for 2 days. Colonies were picked and streaked three times to obtain isolates. Thebacteria were routinely grown either in nutrient agar or nutrient broth. DNA was extractedusing the Quick-DNA fungal/bacterial kits (Zymo Research). The 16S rRNA gene was sequencedusing the primers 27F and 1492R to identify taxonomy (4). The resulting gene sequences werecompared to the 16S rRNA sequence database at NCBI using blastn. Three isolates wereselected for genomic analysis. The 16S rRNA gene from strains G11, G15, and G16 were allgreater than 99% identical to Marinobacter vinifirmus strain FB1, Halomonas alimentaria strainYKJ-16, and Bacillus subtilis strain IAM 12118, respectively. These isolates were Marinobacter sp.strain G11, Halomonas sp. strain G15, and Bacillus sp. strain G16. Whole-genome libraries wereconstructed using the Nextera XT kit (Illumina) and sequenced on the Illumina MiSeq instru-ment using the MiSeq v3 600-cycle kit using a 2  300-bp paired-end sequencing run. Allsequencing libraries were pooled and sequenced on the same MiSeq run.Default parameters were used for all sequence analysis programs unless otherwisenoted. Raw sequencing reads were quality controlled using Trimmomatic version 0.39(5). Nextera adapters were trimmed from the reads. The leading and trailing 3 bp weretrimmed, and the reads were quality filtered using sliding window trimming with awindow size of 4 and quality cutoff phred score of 15. Any reads of less than 36 bp af-ter quality filtering were removed. For G11, 412,903 paired reads survived trimming.For G15, 432,284 paired reads survived trimming. For G16, 816,767 reads survived trim-ming. The trimmed reads were then assembled using SPAdes version 3.15.3 (6) withkmer sizes of 21, 51, 71, 91, 111, and 127. The quality of the assembly was determinedEditor Frank J. Stewart, Montana StateUniversityCopyright © 2022 Techtmann et al. This is anopen-access article distributed under the termsof the Creative Commons Attribution 4.0International license.Address correspondence to Stephen M.Techtmann, smtechtm@mtu.edu.The authors declare no conflict of interest.Received 27 December 2021Accepted 4 February 2022Published 17 February 2022Volume 11 Issue 2 e01232-21 mra.asm.org 1GENOME SEQUENCESDownloaded from https://journals.asm.org/journal/mra on 29 March 2022 by 47.6.27.51.using QUAST version 5.0.2 (7). Annotation of the genomes was performed usingProkka version 1.14.6 (8). Table 1 details the statistics for the genome assemblies andannotation. Antibiotic resistance genes were annotated using the CARD database (9).The genome annotation revealed three resistance nodulation cell division (RND)-type efflux pumps in Marinobacter sp. G11 (two adeF and one rsmA). One RND-typeefflux pump was identified in Halomonas sp. G15 (one rsmA). Two antibiotic inactiva-tion genes and two RND-type efflux pumps were identified in Bacillus sp. G16. Previousstudies have shown that the efflux pumps are important for resistance to glutaralde-hyde (10). This finding suggests that efflux may be a mechanism allowing these bacte-ria to tolerate glutaraldehyde.Data availability. The genome sequences are deposited at GenBank under accessionnumbers JAJSOZ000000000, JAJSPA000000000, and JAJSPB000000000, BioProject numberPRJNA786782, BioSample numbers SAMN23721435, SAMN23721436, and SAMN23721437,and SRA accession numbers SRR17223254, SRR17223255, and SRR17223256.ACKNOWLEDGMENTSThis work was funded by National Science Foundation CBET award 1804685 (MichiganTechnological University) and the Michigan Tech Biological Sciences Department. This workwas performed as part of the Principles of Computational Biology course (BL2700).S.M.T. designed the experiment, analyzed the data, and wrote the announcement.A.L.B. isolated the strains and assisted in sequencing of the genomes. All other authorscontributed to the genome analysis and are listed in alphabetical order by last name.REFERENCES1. Campa MF, Techtmann SM, Gibson CM, Zhu X, Patterson M, Garcia de MatosAmaral A, Ulrich N, Campagna SR, Grant CJ, Lamendella R, Hazen TC. 2018.Impacts of glutaraldehyde on microbial community structure and degrada-tion potential in streams impacted by hydraulic fracturing. Environ Sci Technol52:5989–5999. https://doi.org/10.1021/acs.est.8b00239.2. Kahrilas GA, Blotevogel J, Stewart PS, Borch T. 2015. Biocides in hydraulic frac-turing fluids: a critical review of their usage, mobility, degradation, and toxic-ity. Environ Sci Technol 49:16–32. https://doi.org/10.1021/es503724k.3. Campa MF, Wolfe AK, Techtmann SM, Harik AM, Hazen TC. 2019. Unconven-tional oil and gas energy systems: an unidentified hotspot of antimicrobial re-sistance? Front Microbiol 10:2392. https://doi.org/10.3389/fmicb.2019.02392.4. Frank JA, Reich CI, Sharma S, Weisbaum JS, Wilson BA, Olsen GJ. 2008.Critical evaluation of two primers commonly used for amplification ofbacterial 16S rRNA genes. Appl Environ Microbiol 74:2461–2470. https://doi.org/10.1128/AEM.02272-07.5. Bolger AM, Lohse M, Usadel B. 2014. Trimmomatic: a flexible trimmer forIllumina sequence data. Bioinformatics 30:2114–2120. https://doi.org/10.1093/bioinformatics/btu170.6. Bankevich A, Nurk S, Antipov D, Gurevich AA, Dvorkin M, Kulikov AS, LesinVM, Nikolenko SI, Pham S, Prjibelski AD, Pyshkin AV, Sirotkin AV, Vyahhi N,Tesler G, Alekseyev MA, Pevzner PA. 2012. SPAdes: a new genome assem-bly algorithm and its applications to single-cell sequencing. J ComputBiol 19:455–477. https://doi.org/10.1089/cmb.2012.0021.7. Gurevich A, Saveliev V, Vyahhi N, Tesler G. 2013. QUAST: quality assessmenttool for genome assemblies. Bioinformatics 29:1072–1075. https://doi.org/10.1093/bioinformatics/btt086.8. Seemann T. 2014. Prokka: rapid prokaryotic genome annotation. Bioinfor-matics 30:2068–2069. https://doi.org/10.1093/bioinformatics/btu153.9. Alcock BP, Raphenya AR, Lau TTY, Tsang KK, Bouchard M, Edalatmand A,HuynhW, Nguyen A-LV, Cheng AA, Liu S, Min SY, Miroshnichenko A, Tran H-K,Werfalli RE, Nasir JA, Oloni M, Speicher DJ, Florescu A, Singh B, Faltyn M,Hernandez-Koutoucheva A, Sharma AN, Bordeleau E, Pawlowski AC, ZubykHL, Dooley D, Griffiths E, Maguire F, Winsor GL, Beiko RG, Brinkman FSL, HsiaoWWL, Domselaar GV, McArthur AG. 2020. CARD 2020: antibiotic resistome sur-veillance with the Comprehensive Antibiotic Resistance Database. NucleicAcids Res 48:D517–D525. https://doi.org/10.1093/nar/gkz935.10. Vikram A, Bomberger JM, Bibby KJ. 2015. Efflux as a glutaraldehyde resist-ance mechanism in Pseudomonas fluorescens and Pseudomonas aerugi-nosa biofilms. Antimicrob Agents Chemother 59:3433–3440. https://doi.org/10.1128/AAC.05152-14.TABLE 1 Quality information and features of the de novo assembled strainsFeature Marinobacter sp. G11 Halomonas sp. G15 Bacillus sp. G16GC content (%) 57.9 65.6 43.9Assembly length (bp) 4,040,679 3,811,422 4,258,983Length of longest contig (bp) 288,494 318,971 411,992No. of contigs 126 106 68N50 (bp) 109,800 84,931 259,571L50 12 15 7No. of CDSa 3,791 3,538 4145No. of rRNAs 8 6 13No. of tRNAs 48 71 84a CDS, coding DNA sequences.Techtmann et al.Volume 11 Issue 2 e01232-21 mra.asm.org 2Downloaded from https://journals.asm.org/journal/mra on 29 March 2022 by 47.6.27.51.

Draft Genome Sequencing of Three Glutaraldehyde-Tolerant Bacteria from Produced Water from Hydraulic Fracturing

Draft Genome Sequencing of Three Glutaraldehyde-Tolerant Bacteria from Produced Water from Hydraulic Fracturing

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