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Microbiol Resour Announc
Microbiol Resour Announc
mra
Microbiology Resource Announcements
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American Society for Microbiology 1752 N St., N.W., Washington, DC

39140765
mra00160-24
10.1128/mra.00160-24
mra.00160-24
Genome Sequences
clinical-microbiologyClinical MicrobiologyWhole-genome sequencing of Helicobacter pylori isolates from Native American gastric biopsy specimens
Celona Kimberly 1 Data curation Methodology Writing – original draft Writing – review and editing
Williamson Charles H. D. 1 Data curation Formal analysis Methodology Visualization Writing – original draft Writing – review and editing
Dholakia Rishi 2 Methodology Resources Writing – review and editing
https://orcid.org/0000-0002-2497-0138
Sahl Jason W. 1 3 Methodology Supervision Writing – review and editing
Monroy Fernando P. 3 Funding acquisition Methodology Project administration Resources Supervision Writing – review and editing
https://orcid.org/0000-0003-4331-3683
Settles Erik W. 1 3 Conceptualization Funding acquisition Investigation Project administration Supervision Writing – review and editing Erik.Settles@nau.edu

1 The Pathogen and Microbiome Institute, Northern Arizona University , Flagstaff, Arizona, USA
2 Winslow Indian Health Care Center , Winslow, Arizona, USA
3 Department of Biological Sciences, Northern Arizona University , Flagstaff, Arizona, USA
Editor Putonti Catherine Loyola University Chicago , Chicago, Illinois, USA

Address correspondence to Erik W. Settles, Erik.Settles@nau.edu
The authors declare no conflict of interest.

9 2024
14 8 2024
14 8 2024
13 9 e00160-2416 2 2024
05 7 2024
Copyright © 2024 Celona et al.
2024
Celona et al.
https://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license.

ABSTRACT

Helicobacter pylori infection has been linked to gastrointestinal diseases including gastric cancer. High rates of H. pylori infection and gastric cancer have been reported in indigenous populations within the United States. We report whole-genome sequencing of three H. pylori isolates originating from Native American patients presenting with gastric disease.

KEYWORDS

Helicobacter pylori
gastric cancer
gastrointestinal diseases
Navajo Nation
Native American
HHS | NIH | National Cancer Institute (NCI) 1R21CA248804-01 Monroy Fernando P. Settles Erik W. HHS | NIH | National Cancer Institute (NCI) U54CA143924, U54CA143925 Monroy Fernando P. cover-dateSeptember 2024
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pmcANNOUNCEMENT

Helicobacter pylori, a Gram-negative bacterium, is the causative agent of most human gastric infections and is linked to gastric cancer (1–3). Indigenous communities in the United States have elevated rates of infection and gastric cancer. Members of the Navajo Nation have rates of infection ranging between 56% and 70% (4, 5) and gastric cancer rates three to four times higher than non-Hispanic white populations (6). Navajo patients with gastric symptoms had a gastric biopsy during endoscopy and H. pylori culturing or PCR was performed. H. pylori was present in ~23% of these biopsy samples (7). We describe whole-genome sequencing of three H. pylori isolates from these patients.

Patients were enrolled by informed consent (Navajo Nation IRB #NNR16.263). Gastric pinch biopsy samples were collected during a routine scheduled patient endoscopy. Samples were ground in sterile phosphate-buffered saline, inoculated onto Columbia Agar plates containing 5% defibrinated sheep blood and H. pylori selective supplement (Dent), and incubated for 72 h at 37°C under microaerophilic conditions (5% O2, 10% CO2, and 85% N2). Minimum inhibitory concentrations (MICs) for clarithromycin and metronidazole were determined with ETESTs (bioMérieux) by inoculating 100 µL of a 3 McFarland equivalent isolate suspension onto Mueller-Hinton II plates with 5% defibrinated sheep blood and incubating at 37°C for 4 days under microaerophilic conditions.

Isolate genomic DNA was extracted using a Blood and Tissue Kit (Qiagen) following the manufacturer’s protocol with the additional pretreatment for Gram-negative bacteria. Whole-genome sequencing libraries were generated as previously described (8, 9) except quality was assessed with a Fragment Analyzer using the High Sensitivity NGS fragment kit. Samples were sequenced on the MiSeq platform. Contaminating sequencing reads were identified and removed with the BBsplit tool (BBMap v38.93—sourceforge.net/projects/bbmap/) using phiX (J02482.1) and human (GCF_000001405.39) genomes as references, followed by assignment of taxonomic classifications to reads with kraken2 v2.1.2 (10) and removal of contaminating reads. H. pylori genomes were assembled using SPAdes v3.15.3 (--careful, --cov-cutoff auto) (11). Depth of coverage was calculated from minimap2 v2.24 (-ax sr) (12) alignments using Samtools v1.16.1 (13). Contigs with anomalously low depth of coverage were removed. Assembly metrics were calculated with the statswrapper.sh tool (sourceforge.net/projects/bbmap/ v39.01). Assemblies were annotated with the NCBI Prokaryotic Genome Annotation Pipeline (PGAP) v6.6 (14). Core genome single nucleotide polymorphisms (SNPs) were called from nucmer v3.1 (15) alignments (reference = GCA_017821535.1) within NASP v1.2.1 (16), and a phylogeny was inferred with IQ-TREE v2.2.2.3 (17, 18) from proximity filtered SNPs (distance of 5). The vacA and cagA genotypes were determined with in silico PCR (usearch v11.0.667_i86linux32 – search_pcr, -maxdiffs 2) (19) using previously described primers (20–23). Genomes were screened for antibiotic-resistance markers listed in the Comprehensive Antimicrobial Resistance Database (24).

Genome assembly information is presented in Table 1. The three isolates are putatively genotyped as cagA− and vacA type s2i2m2. A SNP phylogeny (Fig. 1) indicates that the isolates are closely related to isolates originating from Indigenous or Mestizo individuals presenting with gastritis in Mexico (25, 26). ETESTS indicate some isolates are resistant to clarithromycin and metronidazole (Table 1).

TABLE 1 Genome assembly metrics and accession numbers

Isolate	408F-DNA-001	412F-DNA-002	427F-DNA-001	
Assembly accession	JAYXIY000000000	JAYXIX000000000	JAYXIW000000000	
SRA accession	SRR27606653	SRR27606652	SRR27606651	
Sequencing kit	500-cycle Nano v2	500-cycle Nano v2	600-cycle v3	
Sequencing format	2 × 251 bp	2 × 251 bpa	2 × 301 bp	
Total number of paired reads	373,565	441,603	1,201,328	
Average depth of coverage	113×	124×	436×	
Number of contigs	28	19	18	
Genome size (bp)	1,570,731	1,570,272	1,570,218	
L50	4	3	3	
N50 (bp)	176,074	236,910	236,904	
Length of longest contig (bp)	266,682	411,760	411,746	
Average GC content	0.39	0.39	0.39	
Total CDSs (PGAP)	1,502	1,487	1,490	
Minimum inhibitory concentration for clarithromycin (R > 0.25 µg/mL)b	0.125	1.5	0.5	
Minimum inhibitory concentration for metronidazole
(R > 8 µg/mL)c	8	16	1	
Mutations potentially associated with clarithromycin
resistanced	Mutations within 23S rRNA (ARO:3004134) - T510C, G722A, G760del, T896C, T976G, T1024C, C1516del, T1568C, C1648T, T2199C	
Genes/mutations potentially associated with metronidazole resistancee	Presence of major facilitator superfamily antibiotic efflux pump (ARO:3003964); mutations within frxA (ARO:3007059) - V7I, A16T, Q27E, I44V, L71I, F72S, G73S, T110A, N111D, N124S, M126I, A154V, E176K, C193S; mutations within rdxA (ARO:3007055) - T31E, D59N, L62V, S88P, G98S, A118S, V123T, R131K, E175Q	
A Reverse reads trimmed to 228 nucleotides due to sequencing quality.

b Resistance breakpoint for clarithromycin—EUCAST v13.1.

c Resistance breakbpoint for metronidazole—EUCAST v13.1.

d Genomic data queried against features associated with clarithromycin resistance in H. pylori in the Comprehensive Antimicrobial Resistance Database. The mutations in Table 1 were identified within the 23S rRNA gene for all three isolates, but specific mutations listed within the CARD were not identified.

e Genomic data queried against features associated with metronidazole resistance in H. pylori in the Comprehensive Antimicrobial Resistance Database. An antibiotic efflux pump gene was identified in all three genomes. Mutations were identified within frxA and rdxA in all three genomes; specific mutations listed within the CARD are in bold text.

Fig 1 Core genome SNP phylogeny (midpoint rooted) of 186 publicly available H. pylori genomes and three newly sequenced H. pylori genomes. Colors indicate the continent of origin for the H. pylori isolates included in the tree. The blue box highlights the three newly sequenced isolates (408F, 412F, and 427F) and closely related isolates. The three newly sequenced isolates are closely related to isolates collected from Indigenous or Mestizo individuals presenting with gastritis in Mexico.

ACKNOWLEDGMENTS

The authors would like to acknowledge staff and WIHCC (Winslow Indian Health Care Clinic) who recruited and consented these patients to the study. The authors thank Amber Jones for performing whole-genome sequencing.

This work was funded by 1R21CA248804-01, U54CA143925, and U54CA143924.

DATA AVAILABILITY

The whole-genome sequencing project has been deposited under NCBI BioProject PRJNA1066305. Assembly accession numbers and Sequence Read Archive accession numbers are included in Table 1.
==== Refs
REFERENCES

1 Makola D, Peura DA, Crowe SE. 2007 . Helicobacter pylori infection and related gastrointestinal diseases. J Clin Gastroenterol 41 :548–558. doi:10.1097/MCG.0b013e318030e3c3 17577110
2 de Martel C, Ferlay J, Franceschi S, Vignat J, Bray F, Forman D, Plummer M. 2012. Global burden of cancers attributable to infections in 2008: a review and synthetic analysis. Lancet Oncol 13 :607–615. doi:10.1016/S1470-2045(12)70137-7 22575588
3 Eusebi LH, Zagari RM, Bazzoli F. 2014. Epidemiology of Helicobacter pylori infection. Helicobacter 19 Suppl 1 :1–5. doi:10.1111/hel.12165
4 Stancioiu F. 2005. Helicobacter pylori: findings in a native American population. IHS Prim Care Provid 30 :60–63.
5 Harris RB, Brown HE, Begay RL, Sanderson PR, Chief C, Monroy FP, Oren E. 2022. Helicobacter pylori prevalence and risk factors in three rural indigenous communities of northern Arizona. Int J Environ Res Public Health 19 :797. doi:10.3390/ijerph19020797 35055622
6 Anonymous. 2013 Cancer among the Navajo 2005-2013
7 Monroy FP, Brown HE, Sanderson PR, Jarrin G, Mbegbu M, Kyman S, Harris RB. 2022. Helicobacter pylori in native Americans in northern Arizona. Diseases 10 :19. doi:10.3390/diseases10020019 35466189
8 Stone NE, Hall CM, Ortiz M, Hutton SM, Santana-Propper E, Celona KR, Williamson CHD, Bratsch N, Fernandes LGV, Busch JD, Pearson T, Rivera-Garcia S, Soltero F, Galloway R, Sahl JW, Nally JE, Wagner DM. 2022. Diverse lineages of pathogenic leptospira species are widespread in the environment in Puerto Rico. PLoS Negl Trop Dis 16 :e0009959. doi:10.1371/journal.pntd.0009959 35584143
9 Kozarewa I, Turner DJ. 2011. 96-Plex molecular Barcoding for the Illumina genome Analyzer. high-throughput next generation sequencing: Methods and applications 279-298
10 Wood DE, Lu J, Langmead B. 2019. Improved metagenomic analysis with kraken 2. Genome Biol. 20 :257. doi:10.1186/s13059-019-1891-0 31779668
11 Bankevich A, Nurk S, Antipov D, Gurevich AA, Dvorkin M, Kulikov AS, Lesin VM, Nikolenko SI, Pham S, Prjibelski AD, Pyshkin AV, Sirotkin AV, Vyahhi N, Tesler G, Alekseyev MA, Pevzner PA. 2012. SPAdes: a new genome assembly algorithm and its applications to single-cell sequencing. J Comput Biol 19 :455–477. doi:10.1089/cmb.2012.0021 22506599
12 Li H. 2018. Minimap2: pairwise alignment for nucleotide sequences. Bioinformatics 34 :3094–3100. doi:10.1093/bioinformatics/bty191 29750242
13 Li Heng, Handsaker B, Wysoker A, Fennell T, Ruan J, Homer N, Marth G, Abecasis G, Durbin R, 1000 Genome Project Data Processing Subgroup. 2009. The sequence alignment/map format and SAMtools. Bioinformatics 25 :2078–2079. doi:10.1093/bioinformatics/btp352 19505943
14 Tatusova T, DiCuccio M, Badretdin A, Chetvernin V, Nawrocki EP, Zaslavsky L, Lomsadze A, Pruitt KD, Borodovsky M, Ostell J. 2016. NCBI prokaryotic genome annotation pipeline. Nucleic Acids Res. 44 :6614–6624. doi:10.1093/nar/gkw569 27342282
15 Kurtz S, Phillippy A, Delcher AL, Smoot M, Shumway M, Antonescu C, Salzberg SL. 2004. Versatile and open software for comparing large genomes. Genome Biol. 5 :1–9. doi:10.1186/gb-2004-5-2-r12
16 Sahl JW, Lemmer D, Travis J, Schupp JM, Gillece JD, Aziz M, Driebe EM, Drees KP, Hicks ND, Williamson CHD, Hepp CM, Smith DE, Roe C, Engelthaler DM, Wagner DM, Keim P. 2016. NASP: an accurate, rapid method for the identification of SNPs in WGS datasets that supports flexible input and output formats. Microb Genom 2 :e000074. doi:10.1099/mgen.0.000074 28348869
17 Kalyaanamoorthy S, Minh BQ, Wong TKF, von Haeseler A, Jermiin LS. 2017. ModelFinder: fast model selection for accurate phylogenetic estimates. Nat Methods 14 :587–589. doi:10.1038/nmeth.4285 28481363
18 Nguyen L-T, Schmidt HA, von Haeseler A, Minh BQ. 2015. IQ-TREE: a fast and effective stochastic algorithm for estimating maximum-likelihood phylogenies. Mol Biol Evol 32 :268–274. doi:10.1093/molbev/msu300 25371430
19 Edgar RC. 2010. Search and clustering orders of magnitude faster than BLAST. Bioinformatics 26 :2460–2461. doi:10.1093/bioinformatics/btq461 20709691
20 Atherton JC, Cao P, Peek RM, Tummuru MK, Blaser MJ, Cover TL. 1995. Mosaicism in vacuolating cytotoxin alleles of Helicobacter pylori: association of specific vaca types with cytotoxin production and peptic ulceration (∗). J Biol Chem 270 :17771–17777. doi:10.1074/jbc.270.30.17771 7629077
21 Rhead JL, Letley DP, Mohammadi M, Hussein N, Mohagheghi MA, Eshagh Hosseini M, Atherton JC. 2007. A new Helicobacter pylori vacuolating cytotoxin determinant, the intermediate region, is associated with gastric cancer. Gastroenterology 133 :926–936. doi:10.1053/j.gastro.2007.06.056 17854597
22 Yamaoka Y, Kodama T, Gutierrez O, Kim JG, Kashima K, Graham DY. 1999. Relationship between Helicobacter pylori iceA, cagA, and vacA status and clinical outcome: studies in four different countries. J Clin Microbiol 37 :2274–2279. doi:10.1128/JCM.37.7.2274-2279.1999 10364597
23 Yamaoka Y, Kodama T, Kita M, Imanishi J, Kashima K, Graham DY. 1998. Relationship of vacA genotypes of Helicobacter pylori to cagA status, cytotoxin production, and clinical outcome. Helicobacter 3 :241–253. doi:10.1046/j.1523-5378.1998.08056.x 9844065
24 Alcock BP, Huynh W, Chalil R, Smith KW, Raphenya AR, Wlodarski MA, Edalatmand A, Petkau A, Syed SA, Tsang KK, et al. . 2023. CARD 2023: expanded curation, support for machine learning, and resistome prediction at the comprehensive antibiotic resistance database. Nucleic Acids Res 51 :D690–D699. doi:10.1093/nar/gkac920 36263822
25 Camorlinga-Ponce M, Gómez-Delgado A, Aguilar-Zamora E, Torres RC, Giono-Cerezo S, Escobar-Ogaz A, Torres J. 2020. Phenotypic and genotypic antibiotic resistance patterns in Helicobacter pylori strains from ethnically diverse population in Mexico. Front Cell Infect Microbiol 10 :539115. doi:10.3389/fcimb.2020.539115 33643927
26 Muñoz-Ramirez ZY, Pascoe B, Mendez-Tenorio A, Mourkas E, Sandoval-Motta S, Perez-Perez G, Morgan DR, Dominguez RL, Ortiz-Princz D, Cavazza ME, et al. . 2021. A 500-year tale of co-evolution, adaptation, and virulence: Helicobacter pylori in the Americas. ISME J 15 :78–92. doi:10.1038/s41396-020-00758-0 32879462
