Whole Genome Sequences, De Novo Assembly, and Annotation of Antibiotic Resistant Campylobacter jejuni Strains S27, S33, and S36 Newly Isolated from Chicken Meat

Campylobacter is a leading bacterial cause of gastrointestinal infections in humans and has imposed substantial medical and public health burdens worldwide. Among a total of 39 species in the Campylobacter genus, C. jejuni is the most important species responsible for approx. 90% of human Campylobacter illness. Most cases of the infection were acquired by ingesting undercooked poultry meat due to the high prevalence of Campylobacter in the products. Here, we reported the dataset of raw sequences, de novo assembled and annotated genomes of C. jejuni strains S27, S33, and S36 recently isolated from retail chicken by using PacBio highly accurate long-read sequencing technology combined with bioinformatics tools. Our data revealed several virulence and antibiotic resistance genes in each of the chromosomes, a type IV secretion system in the plasmid (pCjS33) of C. jejuni S33, and a type VI secretion system and a phage in the plasmid (pCjS36) of C. jejuni S36. This study not only provides new sequence data but also extends the knowledge pertaining to the genomic and functional aspects of this important foodborne pathogen, including the genetic determinants of virulence and antibiotic resistance.


Introduction
Campylobacter spp.are Gram-negative, spiral-shaped, highly motile bacteria which can cause human diseases such as campylobacteriosis, a form of gastroenteritis characterized by diarrhea, fever, abdominal pain, and nausea [1][2][3].Campylobacter is the most common bacterial cause of foodborne illness in the world, responsible for an estimated 96 million cases annually [4], where 80-90% of human illnesses are due to Campylobacter jejuni [5].The main source of infection is the consumption of contaminated raw or undercooked meat products, particularly poultry [5][6][7] due to the high prevalence of Campylobacter jejuni in retail chicken [8,9].Studies have shown that Campylobacter can colonize the gastrointestinal tract of birds, livestock, and other animals without causing symptoms [10].However, in humans, Campylobacter can invade the intestinal mucosa and trigger an inflammatory response, leading to tissue damage and fluid loss [11].In some cases, Campylobacter infection can also result in serious complications, such as reactive arthritis, Guillain-Barré syndrome, and bacteremia [12].
Given the global burden and public health impact of Campylobacter infection, there is a need for a better understanding of the biology, diversity, and pathogenicity of this bacterium.Whole genome sequencing (WGS) is a powerful tool capable of providing comprehensive information regarding the genetic features and evolutionary relationships of foodborne pathogens such as Campylobacter [13].WGS unveils the presence and distribution of genes associated with virulence, antibiotic resistance, secretion systems, and mobile genetic elements such as phages and plasmids.These elements may influence the survival, adaptation, and virulence of Campylobacter in diverse environments and hosts [14].Furthermore, WGS facilitates the identification and characterization of novel Campylobacter strains, along with detecting and identifying outbreaks and transmission sources [15].Therefore, WGS plays a crucial role in developing more effective and targeted strategies for preventing, diagnosing and treating Campylobacter infection [16].
In this study, the complete genome sequences and annotation of three Campylobacter jejuni strains (S27, S33, and S36) isolated from retail chicken in the United States are presented.An analysis was conducted on their genomic features in comparison to other available C. jejuni strains in public databases addressing their potential implications for food safety and public health.This is the first report of the complete genome sequences and annotation of C. jejuni strains S27, S33, and S36.These findings stand as valuable resources poised to facilitate future studies in comparative and functional genomics of this important foodborne pathogen.

Sample Preparation
C. jejuni strains S27, S33, and S36 were isolated from separate packages of raw chicken samples collected from local supermarkets using a previously described method [17].Briefly, a 450 g chicken sample was rinsed with 250 mL Buffered Peptone Water, BPW (Bio-Rad, Hercules, Ca).The rinse was concentrated by centrifugation and enriched microaerobically (5% O 2 , 10% CO 2 and 85% N 2 ) in Bolton broth (Oxoid, Basingstoke, Hampshire, UK) containing 5% laked horse blood (Remel, Lenexa, KS) and antibiotic selective supplement package containing cefoperazone, trimethoprim, vancomycin, and cycloheximide (Oxoid, Basingstoke, Hampshire, UK) at 42 • C for 24 h.Due to the high motility of Campylobacter, passive filtration with 0.65µm sterile cellulose acetate membranes (Merck-Millipore LTD, Cork Ireland) of the enrichment onto Brucella agar (Becton Dickinson, Sparks, MD) was chosen for bacterial isolation.Following colony purification, the genus and species of the isolates were determined using the previously reported multiplex qPCR assay [18].Genomic DNA extraction was performed using the Qiagen genomic tip 100/G kit (Valencia, CA, USA) and quantified using the Qubit 3.0 fluorimeter (Thermo Fisher Scientific, Waltham, MA, USA) following the manufacturers' instructions.

Whole Genome Sequencing, De Novo Assembly, and Annotation
Whole genome sequencing was conducted in the Sequel II Single Molecule Real Time (SMRT) system (Pacific Biosciences, Menlo Park, CA, USA).The library was constructed using the SMRTbell Prep Kit with the selection of insert sizes ranging from 500 bp to over 20 kb.
The raw reads obtained from PacBio long and accurate HiFi sequencing were deposited in the SRA database in GenBank.Subsequently, they underwent de novo assembly using Canu v2.2 [19] and were trimmed using the "getfasta" command of bedtools software v2.27.1.The sequences were then oriented to the dnaA starting point using Circlator 1.5.5 [20].Functional annotation was carried out using the Rapid Annotation using Subsystem Technology (RAST) server [21].All software was used with default parameters unless otherwise noted.The complete genomes were submitted to the genome database in GenBank and annotated using the NCBI Prokaryotic Genomic Annotation Pipeline (https://www.ncbi.nlm.nih.gov/genome/annotation_prok/,accessed on 7 August 2023) and the Rapid Annotation using Subsystems Technology (RAST, http://rast.nmpdr.org/,accessed on 27 July 2023).

Results
C. jejuni S27, S33, and S36 were isolated from separate packages of fresh chicken collected from local retailers in February 2023.These isolates displayed colonies with round shapes, smooth edges, and a glistening translucent yellowish or pinkish color on Brucellar agar plates.Confirmation of the genus and species of the strains was achieved through a real-time qPCR assay.Whole genome sequencing of C. jejuni S27, S33, and S36 from chicken samples was performed using PacBio long and accurate HiFi sequencing, followed by de novo assembly into complete genomes.Table 1 summarizes the statistics of raw sequence data and assembled complete genomes of the C. jejuni isolates.The assembled genome sizes (~1.6-1.7 Mb) and G + C contents (30.4-30.5%) of these new isolates as shown in Table 1, align closely with other C. jejuni genomes found in the NCBI database.Each strain contains a circular chromosome and, in addition to this, C. jejuni S33 harbors a 40.7 kb plasmid (pCjS33), while C. jejuni S36 carries an 86.8 kb plasmid (pCjS36).Comparison with the available plasmids in GenBank showed that pCjS33 shares 99.30% sequence identity and 96% query coverage with the Campylobacter pTet plasmid, while pCjS36 exhibits up to 99.87% sequence identity and 98% query coverage with multiple C. jejuni plasmids.
The functional prediction of the chromosomes by Rapid Annotation using Subsystems Technology (RAST, http://rast.nmpdr.org/,accessed on 27 July 2023) is summarized in Table 2.Among the subsystems identified by RAST, there were more than 47 genes associated with the virulence, disease causation, defense, and motility of the C. jejuni strains, including cadF, jlpA, porA, and pebA genes associated with adhesion, ciaB, pldA, and flaC for invasion, and a cytolethal distending toxin cdtABC gene cluster.The annotation of the pTet plasmid (pCjS33) in C. jejuni S33 revealed a gene cluster containing the cag pathogenicity island, which encodes a type IV secretion system (T4SS) and a tetO gene that encodes tetracycline resistance protein.This protein protects bacterial ribosomes from binding tetracycline.The prevalence of pTet family plasmids in Campylobacter is notable because they can be horizontally transmitted between the strains, which is a major factor in acquired resistance in Campylobacter spp.In contrast, the plasmid pCjS36 possesses a gene cluster associated with a type VI secretion system (T6SS), which is a novel virulence factor responsible for delivering toxic effectors.These effectors play roles in host colonization, cell adhesion and invasion, and the lysis of erythrocytes [22][23][24].Collectively these findings underscore the potential of the strains to cause human disease.
Furthermore analyses of the C. jejuni S33 and S36 genomes revealed the presence of numerous phage proteins as predicted by the NCBI Prokaryotic Genomic Annotation Pipeline (PGAP) [25].Tables A1 and A2 show the genes and proteins associated with phage packaging, portals, and terminases in C. jejuni S33 and S36, respectively.Consistent with the PGAP predictions, PHASTER, a web-based phage search tool (https://phaster.ca/),predicted an intact 40 kb phage (30.58%GC content) containing 62 proteins along with phage attachment sites (attL and attR) within the sequence region 79591-119622 in C. jejuni S33 chromosome.Similarly, PHASTER detected a 43.4 kb intact phage (29.69%GC content) containing 56 proteins and phage attachment sites (attL and attR) within the sequence region 49017-92474 in C. jejuni S36.Bacteriophage and plasmids play pivotal roles in the horizontal transfer of genetic material.The finding of these mobile genetic elements within the genomes indicates genetic divergence and rearrangement in Campylobacter evolution.

Discussion
Campylobacter jejuni is a major cause of foodborne gastroenteritis worldwide, mainly associated with the consumption of contaminated poultry products.Despite this significance, the complete molecular mechanism behind Campylobacter infections remains incompletely understood, suggesting dependance on a number of virulence factors involving cell adhesion, invasion, and motility [26].Whole genome sequencing was employed to examine the genomic characteristics of three C. jejuni strains (S27, S33, and S36) recently isolated from retail chicken in the United States.Comparing their genomic features with those of other C. jejuni strains available in public databases revealed the presence of virulence determinants within these three strains.These include factors associated with motility (flaAB, flaC, flgE, flgP, flgR, flgS, fliS, fliW, and pflAB) and chemotaxis (cheA, cheW, cheV, cheY, cheR, and cheB), as well as factors associated with adhesion and invasion to host cells (htrA, cadF, flpA, jlpA, capA, porA, pebA, ciaAB, and pldA).Furthermore, these strains carry cytolethal distending toxin (cdtABC) associated with binding to host cells, resulting in enlargement and cell death.Additionally, the strains possess lipooligosaccharide (LOS) facilitating attachment and endocytosis into host cells.
Additionally, a type IV secretion system was identified in the plasmid (pCjS33) of C. jejuni S33, a critical virulence factor typically encoded in mobile genomic islands (plasmids, conjugative elements, or pathogenicity islands).This system is involved in protein transfer across the cell envelope, enhances the oxidative stress response, and contributes to host colonization [27].In contrast, the plasmid (pCjS36) of C. jejuni S36 contains a gene cluster that encodes a type VI secretion system.This system has demonstrated important roles in contact-dependent host cell adherence and invasion, promoting colonization, inducing cytotoxicity of red blood cells, and enhancing survival within the host gastrointestinal tract under conditions of oxidative stress [28,29].
The presence of these genes within the genomes could confer advantages to C. jejuni, enhancing its survival, adaptation, transmission, and pathogenicity across different environments and hosts.These findings emphasize the potential risks associated with C. jejuni infection originating from retail chicken, underscoring the need for improved food safety and public health measures.Moreover, this study contributes to understanding the molecular mechanisms governing C. jejuni-host interactions and horizontal gene transfer.Such insights may facilitate the development of novel therapeutic strategies for managing campylobacteriosis.Notably, this is the first report detailing the complete genome sequences and annotation of C. jejuni strains S27, S33, and S36.These resources hold value for future studies in comparative and functional genomics concerning this important foodborne pathogen.
Author Contributions: Y.H. and J.C.: designing and performing experiments, analyzing data, and writing manuscript.S.K.: bioinformatics analysis and manuscript preparation, S.R. and J.L.: performing experiments.All authors have read and agreed to the published version of the manuscript.

Table 1 .
Statistics of the sequence data and assembled genomes for C. jejuni strains.

Table 2 .
Summarized features of the annotated chromosomes in C. jejuni strains. C.