https://orcid.org/0000-0001-5010-9889
Figures
Abstract
Non-typhoidal Salmonella enterica is a leading cause of gastrointestinal illnesses in the United States. Among the 2,600 different S. enterica serovars, Infantis has been significantly linked to human illnesses and is frequently recovered from broilers and chicken parts in the U.S. A key virulence determinant in serovar Infantis is the presence of the megaplasmid pESI, conferring multidrug resistance. To further characterize the virulence potential of this serovar, the present study identified the types of plasmids harbored by Infantis strains, recovered from surface waters adjacent to leafy greens farms in California. Sequencing analysis showed that each of the examined 12 Infantis strains had a large plasmid ranging in size from 78 kb to 125 kb. In addition, a second 4-kb plasmid was detected in two strains. Plasmid nucleotide queries did not identify the emerging megaplasmid pESI in the examined Infantis strains; however, the detected plasmids each had similarity to a plasmid sequence already cataloged in the nucleotide databases. Subsequent comparative analyses, based on gene presence or absence, divided the detected plasmids into five distinct clusters, and the phylogram revealed these Infantis plasmids were clustered based either on the plasmid conjugation system, IncI and IncF, or on the presence of plasmid phage genes. Assignment of the putative genes to functional categories revealed that the large plasmids contained genes implicated in cell cycle control and division, replication and recombination and defense mechanisms. Further analysis of the mobilome, including prophages and transposons, demonstrated the presence of genes implicated in the release of the bactericidal peptide microcin in the IncF plasmids and identified a Tn10 transposon conferring tetracycline resistance in one of the IncI1 plasmids. These findings indicated that the plasmids in the environmental S. enterica serovar Infantis strains from surface waters harbored a wide variety of genes associated with adaptation, survivability and antimicrobial resistance.
Citation: Quiñones B, Lee BG, Avilés Noriega A, Gorski L (2024) Plasmidome of Salmonella enterica serovar Infantis recovered from surface waters in a major agricultural region for leafy greens in California. PLoS ONE 19(12): e0316466. https://doi.org/10.1371/journal.pone.0316466
Editor: Mabel Kamweli Aworh, North Carolina State University, UNITED STATES OF AMERICA
Received: September 25, 2024; Accepted: December 11, 2024; Published: December 30, 2024
This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.
Data Availability: The annotated nucleotide sequences of the 14 plasmids in the examined S. enterica serovar Infantis strains are found under the GenBank accession numbers PQ365737-PQ365750 (Table 1) in the National Center for Biotechnology Information (NCBI) nucleotide database (https://www.ncbi.nlm.nih.gov/nuccore/), associated with the BioProject accession number PRJNA1162552.
Funding: This work supported by the United States Department of Agriculture, Agricultural Research Service, CRIS Project Numbers 2030-42000-052-00D and 2030-42000-055-00D. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the USDA. USDA is an equal opportunity provider and employer.
Competing interests: The authors have declared that no competing interests exist.
Introduction
Non-typhoidal Salmonella enterica is one of the leading causes of foodborne illness in the United States [1–3]. Salmonellosis is a gastrointestinal illness of mild to moderate severity that can manifest approximately 72 h after exposure to infected food [3, 4]. The species S. enterica contains over 2,600 different serovars, which are differentiated by the combinations of the various somatic O and two phases of flagellar H antigens [5]. However, only about 100 serovars are implicated in most of the illness in humans [6, 7]. Since 2001, there has been a 167% increase in the incidence rate in infections caused by S. enterica serovar Infantis (S. Infantis) [6, 8]. Among the serovars causing human illness, incidence of illness due to S. Infantis moved from the ninth most common in 1996–1998 to the sixth most common in 2019 [9] and since 2016, this serovar has been among the top five isolated from broilers and chicken parts [10]. One factor recognized in the emergence of serovar Infantis strains is the acquisition of a conjugative mega plasmid, termed as plasmid of emerging S. enterica Infantis (pESI) [8, 11, 12]. First described in isolates from Israel in 2014, plasmid pESI was found to harbor multiple antimicrobial resistance genes, including an extended-spectrum-β-lactamase (ESBL) gene [11]. Since 2014, serovar Infantis isolates with this plasmid have been isolated worldwide, primarily from raw poultry; however, it is also present in wildlife and the environment [8, 12, 13]. Multiple drug resistant strains of S. Infantis were reported as early as 1979 in isolates from wild birds in Florida [14].
Plasmids are vectors for transferring resistance, virulence, and adaptation genes in bacteria [15–17]. As mediators of horizontal gene exchange, plasmids carry various types of resistance genes, including antibiotic resistance, metal resistance, and biocide resistance. They also can provide rapid adaptation to an organism during changing environmental conditions [15–17]. In the environment, strains of Salmonella are adept at cycling between host and non-host environments, including surface water, and can tolerate stress and nutrient depletion [18–23]. In several surveys of an important produce-growing region along the Central California coast, Salmonella was routinely isolated, and some strains and subtypes showed indications of persistence and/or regular re-introduction into the environment [24–27]. In one survey of Salmonella from amphibians, reptiles, and surface waters, approximately 62% of the 106 Salmonella strains isolated were resistant to at least one antibiotic [26].
During a 5-year longitudinal survey (2011–2016) of surface waters of the Central California Coastal region, strains of S. Infantis were isolated from 34 samples collected during four of the five years from 13 of the 30 regular sampling sites [27]. Because of the increasing visibility of serovar Infantis strains, and the importance of plasmids in that emergence, the S. Infantis strains from that water survey were studied in depth for the occurrence of plasmids. The aim of this study was to determine plasmid carriage in the strains, and to characterize the types of plasmids among S. Infantis strains in this California water environment.
Materials and methods
Sampling, bacterial culturing, and DNA extraction
The environmental S. Infantis strains used in this study were isolated from surface waters collected during the sampling years 2012 to 2016 [27] (S1 Table). All of the sampling sites were located on public lands within an approximate 20-mile radius of Salinas, California (USA); therefore, no specific permissions were required for the public lands sampling, which did not involve any endangered or protected species. Genomic DNA was extracted from single colonies grown in Trypticase soy broth + 0.6% yeast extract (TSYE, both ingredients from Difco, Franklin Lakes, Nj) using the Wizard Genomic DNA purification kit (Promega, Madison, WI) according to manufacturer’s instructions except that the DNA was resuspended in Buffer EB (Qiagen, Hilden, Germany). Plasmids were extracted as described [28]. Briefly, Salmonella isolates were streaked on TSA agar and incubated overnight at 37°C. Several large colonies were smeared in microcentrifuge tubes and resuspended in lysis buffer followed by incubation at 55°C for 60–90 minutes. Cell lysates were extracted with 100 μl phenol:chloroform:isoamyl alcohol (25:24:1) from which 50–70 μL of DNA were recovered. Samples were electrophoresed on a 0.7–0.8% agarose gel stained with GelRed in 1X Tris Borate EDTA buffer at 4°C for 5 hours at 100V and visualized in a UV imager. Tetracycline resistance was assessed using the Kirby-Bauer method [29] with the BD BBL™ Sensi-Disc™, impregnated with 30 μg of tetracycline (Becton-Dickinson, Franklin Lakes, NJ). Mueller-Hinton agar (Difco) was swabbed with bacterial cultures grown in TSB to an early logarithmic phase. The disks were deposited onto the bacterial lawn, and the plates were further incubated for 18 hours at 37°C. After incubation, the inhibition zones were measured, and the resistance/susceptibility was then determined by following the antimicrobial susceptibility testing guidelines [30].Escherichia coli ATCC strain 25922 was a control for the antibiotic resistance assay.
Library preparation and sequencing
Genomic DNA was prepared using the Wizard Genomic DNA Purification Kit (Promega, Madison, WI). The concentration and quality of the genomic DNA extracted from the recovered Salmonella strains was determined by using a NanoDrop ND-1000 spectrophotometer and a Qubit 4 fluorometer (Thermo Fisher Scientific, Waltham, MA USA). Long-read genome sequencing was performed using a MinION device (Oxford Nanopore Technologies, Oxford, UK). The sequencing libraries were prepared using a total of 200 ng DNA per Salmonella strain using a Rapid Barcoding Sequencing kit SQK-RBK-114.24 (Oxford Nanopore Technologies), following the manufacturer’s workflow recommendations and procedures (https://community.nanoporetech.com/docs/prepare/library_prep_protocols/rapid-sequencing-gdna-barcoding-sqk-rbk114/v/rbk_9176_v114_revm_27nov2022/library-preparation?devices=minion (accessed on 1 March 2024). The prepared libraries were subsequently sequenced on FLO-MIN114 (version 10.4.1 active pore number ≥1100) flow cells (Oxford Nanopore Technologies) for a total of 48 h using MinKNOW software (v24.02.10) with the depletion option (adaptive sampling) using the reference genome from S. Infantis strain CVM N18S1246 (GenBank accession no. CP052777.1) to selectively increase the proportion of plasmid DNA to genomic DNA sequenced [31]. The sequencing reads were base called, trimmed, demultiplexed on super high accuracy mode, filtered on Phred Quality score 8, and saved in the FASTQ format. Subsequently, the error correction and assembly of the sequencing reads were performed with Flye assembler version 2.9 [32] by using the recommended preset options for sequencing reads obtained with the Oxford Nanopore Technologies platform.
To resolve the plasmid sequences in the S. Infantis strains RM18512 and RM18281 into a single contig, the genomes for these strains were sequenced using the Illumina MiSeq System with 500-cycle v2 paired-end sequencing kits (Illumina, San Diego, CA, USA), following the manufacturer’s instructions. DNA libraries were prepared and quantified using the KAPA Low-Throughput Library Preparation Kit and KAPA Library Quantification Kit (Roche, Kapa Biosystems, Wilmington, MA) with modifications as previously described [33]. Libraries were validated with the Agilent 2100 Bioanalyzer chips using the Agilent 12000 kit (Agilent, Santa Clara, CA). The combination of MiSeq and Nanopore sequencing reads were combined by using Unicycler software version v0.4.8 to obtain accurate and complete assemblies of short and long reads [34].
Sequence annotation and comparative analyses
The annotation of the plasmid sequences was performed by using RAST version 2.0 pipeline [11], were visualized using SnapGene software version 7.2 (GSL Biotech LLC, San Diego, CA), and were converted to a five-column tab-delimited annotation file using the GB2Sequin web application prior to submission to GenBank [35]. Confirmation of the Infantis serovar in the examined strains was performed with the SeqSero2 algorithm [36]. To identify the conjugation systems and mobilization families for each plasmid, the amino acid sequences were uploaded to the PlasmidFinder service to determine the mobilization family with MOBscan and to the Galaxy ToolShed site for identifying the conjugation system type with CONJscan [37–40]. To screen the plasmid sequences, the Virulence Finder Database and the Comprehensive Antibiotic Resistance Database [41, 42] were employed to search for genes related to virulence and antimicrobial resistance traits, respectively, and both databases were used in conjunction with the ABRicate tool, version 1.0.1, by selecting the parameter cutoffs of 90% coverage and 95% nucleotide identity [43]. Plasmids also were analyzed with PHASTEST, a web server designed to identify prophage sequences [44]. Furthermore, the comparative analysis of the plasmid sequences was conducted using the Anvi’o platform [45], version 7, by selecting the pangenomics workflow system. The function in the Anvi’o workflow called “anvi-compute-gene-cluster-homogeneity” was employed to compute the geometric and functional homogeneity indexes, and these indexes provided measures of similarity in the detected plasmid genes with an index of 1 being the highest value to indicate no variation. The results obtained using the annotated genomic databases were then subsequently visualized using the command entitled “Anvi-display-pan”. Additionally, the data in the gene cluster summary file from the Anvi’o workflow system were used to analyze the distribution of functional clusters of orthologous gene categories in the examined Salmonella strains. The annotated nucleotide sequences of the 14 plasmids in the examined S. enterica serovar Infantis strains are found under the GenBank accession numbers PQ365737-PQ365750 (Table 1) in the National Center for Biotechnology Information (NCBI) nucleotide database (https://www.ncbi.nlm.nih.gov/nuccore/), associated with the BioProject accession number PRJNA1162552.
Results
Comparative analysis of the non-pESI plasmids in the environmental S. enterica serovar Infantis strains
Initial sequencing analysis showed that 14 strains had a plasmid with sizes ranging from 78 kb to 125 kb (Table 1). The remaining 20 S. Infantis strains did not show bands in the plasmid screening (S1 Table). Twelve strains were selected for subsequent analysis due to high yield recovery of the plasmid DNA. BLASTN searches did not show sequence similarity to the emerging conjugative mega plasmid, pESI; however, each plasmid sequence had some similarity to a plasmid already cataloged in the database (Table 1). A comparative gene analysis was then conducted by employing the Anvi’o workflow to subsequently characterize the plasmid sequences in the examined S. Infantis strains. Phylogenetic analysis, based on gene presence or absence, in the S. Infantis strains divided the plasmid sequences into five distinct grouping (clusters) (Fig 1), and the phylogram revealed that the examined strains were clustered based on the plasmid conjugation system.
The plasmid sequences identified S. enterica Infantis strains were annotated and further compared with the Anvi’o pangenome workflow and visualized with the “Anvi-display-pan” command.
Moreover, further analysis of the contigs revealed a small multicopy 4 kb-plasmid in strains RM17050 and RM16752, which was assigned to cluster I, and displayed 100% identity (99% coverage) to plasmid pST46-2 from S. enterica serovar Typhimurium (Fig 1 and Table 1). The IncI1-based conjugation system, belonging to the MOBP family, was identified in cluster II plasmids pRM16752-1, pRM17050-1, pRM18147, pRM18148 and pRM18410. Plasmids pRM17050-1 (86% coverage) and pRM16752-1 (99% coverage) showed 95–99% similarity, respectively, to pK2721-1 from Citrobacter koseri strain (Table 1). A high similarity of 99% was also observed for plasmids pRM18148 and pRM18410 with the clinical Escherichia coli plasmid pEC00592_5 (100% coverage) and the chicken meat E. coli plasmid pCH56 (94% coverage), respectively. Cluster II plasmid pRM18147 showed 98% similarity (96% coverage) with plasmid pWP2-W18-CRE-03_2 from E. coli (Table 1), recovered from wastewater.
The IncF-based conjugation system, belonging to the MOBF family, was detected in plasmids pRM18512, pRM18281 and pRM21082. However, cluster IV plasmid pRM21082, displaying 100% similarity (96% coverage) to plasmid pCFSAN058598_01, was only identified in strain RM21082 and was found to be 3000 bp smaller than the plasmids pRM18512 and pRM18281, which were assigned to cluster V and showed sequence similarity to plasmid pRM11060-2 (100% identity; 40% coverage) (Table 1). Moreover, plasmids pRM17884, pRM17885, pRM21071 and pRM21154, assigned to cluster III, did not contain a conjugation system (Table 1); however, further analysis of the sequences with PHASTEST revealed that the cluster III plasmids resembled a phage plasmid, and showed the closest sequence similarity (99% identity; 88–99% coverage) to a plasmid found in the clinical strain, S. enterica 2013K-1747 (Table 1). Plasmid pRM18418 showed 99% sequence similarity to plasmid pN18S0935-1 (98% coverage) in S. serovar Kentucky strain N18S0935 and to plasmid pN17S166 (92% coverage) recovered from S. enterica serovar 4, [5],12:i:− strain CVM N17S166.
As further demonstrated in the comparative analysis with the Anvi’o workflow (Fig 1), various homogeneity indexes were determined to measure the variation and frequency of their occurrence in the detected plasmid genes with an index of 1 being the highest value indicating no variation. In this study, the average homogeneity index value was determined to be 0.95 to 0.96 for all plasmid sequences combined. Subsequent measurement of the geometric and functional homogeneity indexes was performed. In particular, the geometric homogeneity index determined the similarity between gene variations based on how many gaps exist between the different sequences in the gene. The functional homogeneity index measured the similarity between gene variations based on how many differences exist between aligned sequences of the gene variations. The results obtained in this study identified notable genes, linked to plasmid stability, segregation and replication, with lower geometric homogeneity such as the DNA methylase subunit (HsdM) in the IncI1 plasmids with an index of 0.64, the chromosome segregation protein (Spo0J) in the IncF plasmids with an index of 0.65, and the DNA polymerase (DinB) with an index of 0.64. Additionally, other identified genes, associated with plasmid transferability and replication, with lower functional homogeneity were the single-stranded DNA-binding protein (Ssb) among the IncF and IncI1 plasmids with an index of 0.80, the type IV secretory pathway component (VirB4) among the IncF plasmids with an index of 0.71, and the SOS-response transcriptional repressor (LexA) among the IncF and IncI1 plasmids with an index of 0.81.
Characterization of the functional gene categories in the examined plasmids from S. enterica serovar Infantis strains
Further analysis of the detected genes in the Infantis plasmids revealed that on average 25% of the genes in all of the examined plasmids were assigned to functional categories (Table 1). In particular, the two small 4 kb-plasmids in cluster I had the highest proportion of assigned categories. The cluster II and cluster III plasmids with an average plasmid size of 85.4 kb to 89.4 kb had about 20–23% of detected genes with an assigned function. The largest plasmids in clusters IV and V with an average size of 91.5 kb and 121 kb, respectively, were found to have a higher proportion of the genes (38%) assigned to functional categories (Table 1). By conducting a subsequent analysis with the Anvi’o workflow, the presence of putative genes for strains belonging to each grouping was identified, and the genes assigned to specific functional categories were further examined (Fig 2). All the large plasmids (>78 Kb) contained at least 4% of genes in the functional category for cell cycle control and cell division (D category), replication and recombination (L category) as well as defense mechanisms and transcription (V and K categories, respectively).
The assignment to functional clusters of orthologous genes categories was determined from the gene cluster summary file using the Anvi’o workflow for the examined S. Infantis plasmids, grouped in various clusters.
Subsequent analysis of cell cycle control and division functional category revealed the presence in the large plasmids of parA and paB-like genes, involved in plasmid division. Examination of genes involved in replication, recombination and repair demonstrated the large plasmids contained a site-specific recombinase xerD like gene. The phage-containing plasmids in cluster III and IncF plasmids in cluster II (p91487, p116460, and p126613) contained an additional site-specific recombinase like gene resembling pinE. These phage-containing plasmids also harbor a DNA methylase-like gene and a gene resembling a DNA recombination-dependent growth factor RdgC. The remaining plasmids had a different DNA methylase yhdj identified, as well as genes supporting DNA repair.
To characterize genes associated with defense mechanisms, cluster III plasmids were found to contain higA and higB like genes, encoding the HigAB toxin antitoxin module for plasmid maintenance. The IncI1 plasmids, including pRM18148, contained the ardA antirestriction gene. The IncF plasmids contained a type I restriction system as well as an emrAB coding for an efflux pump system.
On the subsequent analysis of genes involved in transcription, all large plasmids contained a form of a SOS-response transcriptional repressor LexA. In particular, plasmids pRM18512 and pRM18281 contained a predicted transcriptional regulator with a different ribbon-helix-helix (RHH_1) domain and predicted DNA-binding transcriptional regulator. Plasmid pRM21082 contained a predicted RNA polymerase-binding transcription factor DksA, and a putative transcriptional regulator GlxA. Most IncI1 plasmids, with the exception of pRM18148, contained a predicted putA1 ribbon-helix-helix transcription regulator while plasmid pRM18148 alternately contained predicted DNA-binding proteins in the AraC and AcrA family. On the analysis of cell motility, the IncI1 plasmids contained the effector genes, pilK-pilV associated with the type II secretion system, including a pseudopilin, resembling a flagellum. The IncF plasmids contained the RNA binding protein ProQ, involved in post-translational regulation.
Examination of the mobilome functional category X, including prophages and transposons, showed that cluster III plasmids contained genes involved in regulation and structure of the phage. Additional observations revealed the presence of genes with a lytic trans-glycosylase lysM domain, assigned to biogenesis of the cell wall and membrane category M (Fig 2). Also, functional genes associated with signal transduction mechanisms (category T) included the cpdA gene coding for 3’,5’-cyclic AMP phosphodiesterase in the cluster III plasmids and the sRNA binding gene proQ in the cluster V IncF plasmids. Moreover, the IncF plasmids pRM18512 and pRM18281 contained a transposase system in the mobilome functional category that encoded microcin V (mccV) as well as a predicted arabinose efflux permease araJ like gene (category G) (Fig 2). The IncF plasmids also contained the macrolide efflux protein A MefA as part of a major facilitator superfamily involved in substrate transport across membranes. Only the IncF plasmid pRM21082 harbored a pilus assembly gene fimC encoding for a chaperone identified in the functional category assigned to extracellular structures (category W).
Tetracycline resistance determinants are present in the S. Infantis plasmid pRM18148
By using a validated database to screen for antimicrobial resistance genes, the analysis revealed that only one of the examined Infantis plasmids, the IncI plasmid pRM18148 had genes conferring tetracycline resistance (Fig 3). Phenotypic assays employing the disk diffusion method corroborated in strain RM18148 a strong resistance based on the absence of a zone of inhibition surrounding the tetracycline-infused disk. Further analysis with the TnCentral web portal of the region conferring tetracycline resistance in the Infantis plasmid pRM18148 indicated that these antimicrobial resistance genes were contained within a partial sequence of the Tn10 transposon (Fig 3), a composite mobile element which was also previously found in an unnamed plasmid identified in S. enterica serovar Heidelberg strain FDAARGOS_319 (Accession No. CP027411). Analysis of the coding sequences in the Infantis plasmid pRM18148 confirmed the presence of the TetB efflux pump, flanked by the tetracycline resistance transcriptional repressor TetR, the tetracyline resistance-associated transcriptional repressor TetC, and the multidrug resistant efflux pump AcrAB transcriptional activator RobA.
The RAST annotated nucleotide sequences for plasmid pRM18148 were uploaded to SnapGene software, version 7.2 (GSL Biotech LLC, San Diego, CA) to generate the plasmid map.
One difference identified in the Infantis plasmid pRM18148 was the presence of a putative adenosylcobinamide-phosphate synthase (cbiB) gene (Fig 3), a region with a potential role in conjugation and coenzyme B12 biosynthesis. Other notable features identified in the Infantis plasmid pRM18148 included the SOS-inhibitors PsiA and PsiB, a shufflon-specific DNA recombinase, and the antirestriction protein ArdA (Fig 3). On the identification of the conjugal apparatus, plasmid pRM18148 was found to harbor the traABC region as well as the relaxase protein NikB. Other notable genes needed for conjugation were a shufflon-specific DNA recombinase, and the ardA gene, and a pil gene cluster required for Type IV pilus formation.
Discussion
S. enterica serovar Infantis is a clinically relevant serovar, and recent epidemiological studies have indicated a 167% increase in human infections in the United States [6, 8]. The unique characteristics of S. Infantis as a critical human pathogen is strongly correlated with the presence of plasmid pESI, providing resistance to multiple antibiotic classes as well as an enhanced biofilm formation, adhesion and invasion into host mammalian cells [11]. To further characterize the potential emergence of S. Infantis in agricultural regions for fresh produce in Central California, the objective of the present study was to determine the occurrence of plasmids, potentially conferring multidrug resistance in serovar Infantis strains, previously recovered from a survey of surface waters [27]. Although the analysis of the plasmid carriage failed to identify the multidrug resistant plasmid pESI in the recovered the S. Infantis strains from surface waters, the comparative sequence analysis of the detected plasmids with critically important conjugation groups IncI1 and IncF. In particular, IncI1 plasmids are a common plasmid type among the Enterobacteriaceae and have been isolated from food and clinical sources. Because of their potential to carry antibiotic resistance and virulence genes, IncI1 plasmids are a cause of public health concern [46]. Studies with E. coli and Salmonella 4,5,12:i:− strains showed that carriage of the IncI1 plasmid carried no measurable fitness cost on the bacteria in terms of association and invasion in vitro [46, 47]. IncF plasmids are also known for transferring antibiotic resistance determinants. The IncF family may contain SpvB, an ADP-ribosylating toxin, which allows it to cause systemic infection in humans. To date, the SvpB-IncF plasmid has been found in nine different Salmonella serovars, not including Infantis [48–50]. The pESI plasmid is a member of the IncF1B family [12].
Sequence comparison of the examined Infantis plasmids with those deposited in the databases indicated that the plasmids from this study showed significant sequence similarity to plasmids previously identified both locally and across the globe and indicated the ability of these plasmid types to move among the microbial population. In particular, the cluster I plasmids showed the closest similarity to a plasmid sequenced from a multidrug resistant S. enterica serovar Typhimurium strain 46 isolated from a clinical sample in China [51]. Interestingly, the cluster V plasmids were most similar to plasmid pRM11060-2, previously described from a different Salmonella strain with the serotype 6,8:d:− recovered from surface waters collected in Central California in 2009 [52]. When compared to plasmids grouped in cluster V, plasmid pRM11060-2 contained an additional 154-kb fragment encoding a prophage and hemolysin [52]. The S. Infantis strains RM18512 and RM18281 were isolated from water samples collected in 2013–2014 in the same agricultural region [27]. These observations suggested that the IncF plasmid, detected in Infantis and 6,8:d:− strains isolated 4–5 years apart, appeared to be prevalent in this surveyed agricultural region in Central California and are regularly re-introduced and move among different strains and serotypes of Salmonella. Consistent with this hypothesis, further analysis of the homogeneity indexes indicated that a high degree of gene variability was identified in relevant traits related to plasmid segregation, conjugation, replication, and stability. Variability in these among all the plasmid genes could potentially have an effect on the ability of these plasmids to move determinants among different genera of bacteria or different hosts within a genus in the environment, and this variability could subsequently influence the bacterial host virulence, adaptation and fitness to environments [16, 17, 50]. The closest similarities of the IncI1 plasmid pRM18148 were reported in Salmonella serovars Kentucky and the monophasic 4, [5],12:i:-. The Infantis plasmid carried the cdiB gene, which encodes an integral membrane protein important for the last step in Vitamin B12 synthesis in Salmonella serovar Typhimurium [53].
Moreover, the present study also documented the presence of phage plasmids, a plasmid type with a potential for both horizontal and vertical transfers [54]. Phage plasmids have been described previously in Salmonella [54–56], and this type of mobilizable genetic element is capable of conferring extended-spectrum β-lactamase resistance in S. enterica serovar Typhi and other bacteria [54, 57]. In the present study, the phage plasmids in cluster III showed sequence similarity to a plasmid from the clinical S. enterica strain 2013K-1747. Although there is limited published information about location, transmission vehicle and illness caused by strain 2013K-1747, the genome sequence was included as part of a larger study of genomic typing schemes for Salmonella [58]. Subsequent genome sequence analysis, using SeqSero2 algorithm [36], revealed strain 2013K-1747 to be a S. enterica serovar Adalaide. The appearance of very similar phage plasmids in two serovars, Infantis and Adalaide, indicate movement of this type of plasmid among Salmonella strains.
The presence of cell cycle control genes is to be expected among plasmids that are maintained in populations, particularly those genes that control replication, recombination, and repair. The ParABS DNA partitioning system was originally described in low copy number plasmids, like those described here [59, 60]. Similarly, the large plasmids contained a gene similar to xerD, which is involved in the transfer of genetic information, specifically the resolution of plasmid multimers and integration of DNA elements into host genomes [61]. This system allows for an extension of the host range of the plasmids and may play a role in the movement of these plasmids between strains and between genera.
Although no virulence factors were initially detected according to the output provided by the Virulence Finder Database [42], subsequent analysis of the functional gene categories in all of the S. Infantis plasmids identified several pathogenesis determinants conferring Salmonella an ability to potentially adhere, invade as well as colonize and survive in the host mammalian cells. In particular, the HigAB toxin-antitoxin module, present in the Cluster III plasmids, is involved in plasmid maintenance. This marker can also alter the production of virulence factors, as previously shown for another human pathogen, Pseudomonas aeruginosa [62]. The toxin-antitoxin systems were first described in F-plasmids and work with toxin genes present on the chromosome to allow for the survival of cells only if they retain the plasmid encoding the cognate antitoxin. These systems have been described in several different species of bacteria, including human pathogenic bacteria, and can be involved in virulence, stress response, and protein secretion [63, 64]. Other pathogenesis determinants include the ardA gene present in the IncI1 plasmids with a function in evading host Type I DNA restriction systems, and allowing plasmid-borne virulence genes to be maintained and spread [65]. The emrAB efflux coding system, which was present in the IncF plasmids, plays roles in both antibiotic and chromate resistance, and has been found in other human pathogenic bacteria isolated from the environment [66, 67]. The dksA gene, which was present in the IncF plasmid pRM21082, plays a role in redox-based signaling in response to nitrosative and oxidative stress in Salmonella, and control of these defenses contributes to Salmonella pathogenesis [68]. The pilK-pilV genes, which were present in the IncI1 plasmids, are expressed in Salmonella Typhi and are thought to play a role in the virulence of that pathogen. In other Salmonella isolates, the pil genes on the IncI1 R64 plasmid encode the Type IVb pilus for conjugation [69]. Mutants in pilS, the major pilin subunit, had reduced adhesion and invasion of human epithelial gastrointestinal cells in vitro [70, 71].
Additional genes that may have roles in pathogenesis included mccV in the mobilome of the Cluster V, IncF plasmids, which encodes microcin V, a low molecular weight antibiotic involved in competitive interactions among species belonging to Enterobacteriaceae and with a role in promoting a successful colonization and survival in host epithelial cells [72, 73]. These same plasmids also contained the arabinose efflux permease araJ, which was previously implicated as an inducer of biofilm production in S. Typhimurium [74]. Additionally, the presence of the macrolide efflux protein A MefA, a member of the major facilitator superfamily [75], may confer an increased ability for the uptake of nutrients and extrusion of harmful compounds such as antibiotics and heavy metals [75, 76]. Moreover, the expression of the ProQ RNA binding protein in these IncF plasmids regulates the expression of virulence gene repertoire, promoting Salmonella intracellular growth during host cell colonization [77].
In the analysis of functional gene categories with a role in the biogenesis of the cell wall and membrane with a role in virulence, the S. Infantis cluster III phage plasmids harbored genes with a lytic trans-glycosylase LysM domain, proposed to be involved in a wide array of functions such as the routine maintenance of the peptidoglycan structure, engaging in bacterial resistance against the action of β-lactam antibiotics, and virulence pathways during host invasion [78]. Genes implicated in signal transduction mechanisms in the cluster III plasmids included the expression of 3′,5′-cAMP phosphodiesterase, required for fine-tuning the intracellular levels of the global virulence regulator cAMP in bacterial pathogens [79].
The use of the Comprehensive Antibiotic Resistance Database [41] in the examined S. Infantis strains revealed that only the IncI1 plasmid pRM18148 carried an antibiotic resistance marker. Using the TnCentral web portal [80], markers conferring resistance to tetracycline were found within a partial sequence of the Tn10 transposon, and tetracyline resistance in strain RM18148 was corroborated phenotypically in this study. The Tn10 transposon is a well-characterized composite element with the ability to move between other plasmids and host genomes [81], and this composite mobile element has been previously documented in S. enterica serovar Heidelberg strain FDAARGOS_319 (Accession No. CP027411) [82]. Tetracylines are widely used agents in agricultural settings [83], and resistance to these agents has been one of the most commonly identified in the plasmids among S. enterica isolates from food animals in the United States [84], highlighting the need for better monitoring on the use of these agents in agriculture. Additionally, pRM18148 is an IncI1 plasmid and carries the necessary coding for conjugation, indicating that the tetracycline resistance cassette could readily move between Salmonella strains in the environment [46]. The traABC region and the nikB gene encode proteins needed for the formation of the relaxosome to initiate transfer of plasmid DNA [46]. The antirestriction protein ArdA functions in bacterial conjugation to allow an unmodified plasmid to evade restriction modification system in the recipient host [65, 85].
In summary, the present study employed high-resolution sequencing to characterize mobilizable plasmids, belonging to different incompatibility groups, in S. enterica serovar Infantis strains, recovered from surface waters adjacent to leafy greens farms in California. The comparative analysis of functional genes led to the identification of pathogenesis and antimicrobial resistance determinants that can contribute to the virulence potential of the S. Infantis strains from an important agricultural region.
Conclusions
To further characterize plasmid carriage, the objective of the present study was to examine the plasmidome among strains of S. enterica serovar Infantis, recovered from surface waters proximal to leafy greens fields in Central California, an important agricultural region in the United States. Among the Salmonella serovars, Infantis continues to contribute significantly to the incidence of human infections worldwide due to the presence of the megaplasmid pESI. Although the plasmid pESI was not identified in the examined S. Infantis in this study, mobilizable IncI1, IncF, and phage plasmids were detected that were harboring determinants implicated in virulence and antimicrobial resistance. The analysis of functional gene categories of the annotated sequences in the Infantis plasmids revealed determinants with roles implicated in promoting the successful colonization and survival of host by enhancing the adhesion and invasion of epithelial cells and by increasing the nutrient uptake and the extrusion of harmful compounds and antimicrobials. Additionally, the identification of the composite Tn10 transposon, conferring tetracycline resistance in strain RM18148, underline the necessity of better monitoring of commonly used agents in agriculture. Moreover, the findings from this study provided evidence for IncF plasmid movement among different Salmonella serovars, recovered from surface waters in Central California. The high similarity of the Infantis IncF plasmids to plasmid pRM11060-2 from a different Salmonella with the serotype 6,8:d:−, isolated in a previous surface water survey, demonstrated that these plasmids continue to be prevalent among strains and serovars of S. enterica in this agricultural region for leafy greens. This study has provided fundamental information based on annotated sequence data, and additional comparative and functional analyses are thus needed to determine the expression patterns of the identified genes and dissect how their expression contributes to the virulence phenotypes. Leafy greens have been previously a vehicle of foodborne illness, and surface waters can be a potential a route of transmission of bacterial pathogens; therefore, subsequent phenotypical studies are aimed at further categorizing the virulence potential as well as the environmental fitness, adaptability and persistence of the examined S. Infantis strains, recovered from this important agricultural region for fresh produce.
Supporting information
S1 Table. Salmonella enterica serovar Infantis strains screened for plasmids in this study.
https://doi.org/10.1371/journal.pone.0316466.s001
(DOCX)
Acknowledgments
The authors thank Dr. Dayna Harhay (USDA-ARS, U.S. Meat Animal Research Center, Clay Center, NE) for providing technical advice and E. coli strains harboring control plasmids.
References
- 1. Dewey-Mattia D, Manikonda K, Hall AJ, Wise ME, Crowe SJ. Surveillance for foodborne disease outbreaks—United States, 2009–2015. MMWR Surveill Summ. 2018;67(10):1–11. pmid:30048426
- 2. U.S. Centers for Disease Control. FoodNet Fast pathogen surveillance. 2023 [cited 5 September 2023]. Available from: https://wwwn.cdc.gov/foodnetfast/.
- 3. Scallan Walter EJ, Griffin PM, Bruce BB, Hoekstra RM. Estimating the number of illnesses caused by agents transmitted commonly through food: A Scoping Review. Foodborne pathogens and disease. 2021;18(12):841–58. pmid:34529512
- 4. U.S. Centers for Disease Control and Prevention. Salmonella. 2024 [cited 1 October 2024]. Available from: https://www.cdc.gov/salmonella/outbreaks/index.html.
- 5.
Grimont PAD, Weill F-X. Antigenic formulae of the Salmonella serovars, 9th edition. Paris, France: Pasteur Institute: World Health Organization Collaborating Centre for Reference and Research on Salmonella; 2007.
- 6. U.S. Centers for Disease Control and Prevention. National enteric disease surveillance: Salmonella annual report, 2016. 2018 [cited 8 July 2024]. Available from: https://stacks.cdc.gov/view/cdc/58450.
- 7. European Food Safety Authority. The European Union One Health 2021 Zoonoses Report. EFSA J. 2022;20(12):e07666. pmid:36524203
- 8. Mattock J, Chattaway MA, Hartman H, Dallman TJ, Smith AM, Keddy K, et al. A One Health perspective on Salmonella enterica serovar Infantis, an emerging human multidrug-resistant pathogen. Emerging infectious diseases. 2024;30(4):701–10. https://doi.org10.3201/eid3004.231031
- 9. Tack DM, Ray L, Griffin PM, Cieslak PR, Dunn J, Rissman T, et al. Preliminary incidence and trends of infections with pathogens transmitted commonly through food—Foodborne Diseases Active Surveillance Network, 10 U.S. sites, 2016–2019. Morb Mortal Wkly Rep. 2020;69(17):509–14. https://doi.org10.15585/mmwr.mm6917a1
- 10.
U.S. Department of Agriculture Food Safety Inspection Service. Quarterly sampling reports on Salmonella and Campylobacter. 2024 [cited 9 November 2024]. Available from: https://www.fsis.usda.gov/science-data/data-sets-visualizations/microbiology/microbiological-testing-program-rte-meat-and-7.
- 11. Aviv G, Tsyba K, Steck N, Salmon-Divon M, Cornelius A, Rahav G, et al. A unique megaplasmid contributes to stress tolerance and pathogenicity of an emergent Salmonella enterica serovar Infantis strain. Environ Microbiol. 2014;16(4):977–94. https://doi.org10.1111/1462-2920.12351
- 12. Alvarez DM, Barrón-Montenegro R, Conejeros J, Rivera D, Undurraga EA, Moreno-Switt AI. A review of the global emergence of multidrug-resistant Salmonella enterica subsp. enterica serovar Infantis. Intl J Food Microbiol. 2023;403:110297. https://doi.org10.1016/j.ijfoodmicro.2023.110297
- 13. McMillan EA, Weinroth MD, Frye JG. Increased prevalence of Salmonella Infantis isolated from raw chicken and turkey products in the United States is due to a single clonal lineage carrying the pESI plasmid. Microorganisms. 2022;10(7):1478. https://doi.org10.3390/microorganisms10071478
- 14. White FH, Forrester DJ. Antimicrobial resistant Salmonella spp. isolated from double-crested cormorants (Phalocrocorax auritus) and common loons (Gavia immer) in Florida. J Wildlife Dis. 1979;15(2):235–7. https://doi.org10.7589/0090-3558-15.2.235
- 15. Smalla K, Jechalke S, Top EM. Plasmid detection, characterization, and ecology. Microbiol Spectr. 2015;3(1):PLAS-0038-2014. pmid:26104560
- 16. Rodríguez-Beltrán J, DelaFuente J, León-Sampedro R, MacLean RC, San Millán Á. Beyond horizontal gene transfer: The role of plasmids in bacterial evolution. Nat Rev Microbiol 2021;19(6):347–59. pmid:33469168
- 17. Wein T, Dagan T. Plasmid evolution. Current biology: CB. 2020;30(19):R1158–R63. https://doi.org10.1016/j.cub.2020.07.003
- 18. Winfield MD, Groisman EA. Role of nonhost environments in the lifestyles of Salmonella and Escherichia coli. Appl Environ Microbiol 2003;69(7):3687–94. https://doi.org10.1128/AEM.69.7.3687-3694.2003
- 19. Liu H, Whitehouse CA, Li B. Presence and persistence of Salmonella in water: the impact on microbial quality of water and food safety. Front Public Health. 2018;6:159. https://doi.org10.3389/fpubh.2018.00159
- 20. Topalcengiz Z, McEgan R, Danyluk MD. Fate of Salmonella in Central Florida surface waters and evaluation of EPA worst case water as a standard medium. J Food Prot. 2019;82(6):916–25. https://doi.org10.4315/0362-028X.JFP-18-331
- 21. Thomason BM, Biddle JW, Cherry WB. Detection of Salmonellae in the environment. Appl Microbiol. 1975;30(5):764–7. https://doi.org10.1128/am.30.5.764-767.1975
- 22. Parker CT, Huynh S, Quiñones B, Harris LJ, Mandrell RE. Comparison of genotypes of Salmonella enterica serovar Enteritidis phage type 30 and 9c strains isolated during three outbreaks associated with raw almonds. Appl Environ Microbiol 2010;76(11):3723–31.
- 23. Fatica MK, Schneider KR. Salmonella and produce: survival in the plant environment and implications in food safety. Virulence. 2011;2:573–9. https://doi.org10.4161/viru.2.6.17880
- 24. Cooley MB, Quiñones B, Oryang D, Mandrell RE, Gorski L. Prevalence of Shiga toxin-producing Escherichia coli, Salmonella enterica, and Listeria monocytogenes at public access watershed sites in a California Central Coast agricultural region. Front Cell Infect Microbiol. 2014;4:30. https://doi.org10.3389/fcimb.2014.00030
- 25. Gorski L, Parker CT, Liang A, Cooley MB, Jay-Russell MT, Gordus A, et al. Prevalence, distribution, and diversity of Salmonella enterica in a major produce region of California. Appl Environ Microbiol 2011;77(8):2734–48. https://doi.org10.1128/AEM.02321-10
- 26. Gorski L, Jay-Russell MT, Liang AS, Walker S, Bengson Y, Govoni J, et al. Diversity of pulsed field gel electrophoresis pulsotypes, serovars and antibiotic resistance among Salmonella isolates from wild amphibians and reptiles in the California Central Coast. Foodborne Path Dis. 2013;10:540–8. https://doi.org10.1089/fpd.2012.1372
- 27. Gorski L, Liang AS, Walker S, Carychao D, Aviles Noriega A, Mandrell RE, et al. Salmonella enterica serovar diversity, distribution, and prevalence in public-access waters from a Central California coastal leafy green-growing region from 2011 to 2016. Appl Environ Microbiol 2022;88:e01834–21. https://doi.org10.1128/AEM.01834-21
- 28. Kado CI, Liu ST. Rapid procedure for detection and isolation of large and small plasmids. J Bacteriol. 1981;145(3):1365–73. pmid:7009583
- 29. Hudzicki J. Kirby-Bauer disk diffusion susceptibility test protocol. 2009 [cited 23 January 2023]. Available from: https://asm.org/protocols/kirby-bauer-disk-diffusion-susceptibility-test-pro.
- 30.
Clinical and Laboratory Standards Institute (CLSI). Performance Standards for Antimicrobial Susceptibility Testing 34th Ed. Clinical and Laboratory Standards Institute, USA: CLSI Document M100; 2024.
- 31. Ulrich JU, Epping L, Pilz T, Walther B, Stingl K, Semmler T, et al. Nanopore adaptive sampling effectively enriches bacterial plasmids. mSystems. 2024;9(3):e0094523. pmid:38376263
- 32. Kolmogorov M, Yuan J, Lin Y, Pevzner PA. Assembly of long, error-prone reads using repeat graphs. Nat Biotechnol 2019;37(5):540–6. pmid:30936562
- 33. Parker CT, Huynh S, Alexander A, Oliver AS, Cooper KK. Genomic characterization of Salmonella typhimurium DT104 Strains associated with cattle and beef products. Pathogens. 2021;10(5):529. https://doi.org10.3390/pathogens10050529
- 34. Wick RR, Judd LM, Gorrie CL, Holt KE. Unicycler: Resolving bacterial genome assemblies from short and long sequencing reads. PLoS Comput Biol. 2017;13(6):e1005595. pmid:28594827
- 35. Lehwark P, Greiner S. GB2sequin—A file converter preparing custom GenBank files for database submission. Genomics. 2019;111(4):759–61. pmid:29842948
- 36. Zhang S, den Bakker Hendrik C, Li S, Chen J, Dinsmore Blake A, Lane C, et al. SeqSero2: Rapid and improved Salmonella serotype determination using whole-genome sequencing data. Appl Environ Microbiol 2019;85(23):e01746–19. https://doi.org10.1128/AEM.01746-19
- 37. Blankenberg D, Von Kuster G, Bouvier E, Baker D, Afgan E, Stoler N, et al. Dissemination of scientific software with Galaxy ToolShed. Genome Biol. 2014;15(2):403. pmid:25001293
- 38.
Carattoli A, Hasman H. PlasmidFinder and In Silico pMLST: Identification and Typing of Plasmid Replicons in Whole-Genome Sequencing (WGS). In: de la Cruz F, editor. Horizontal Gene Transfer: Methods and Protocols. New York, NY: Springer US; 2020. p. 285–94.
- 39. Cury J, Abby SS, Doppelt-Azeroual O, Néron B, Rocha EPC. Identifying conjugative plasmids and integrative conjugative elements with CONJscan. In: de la Cruz F, editor. Horizontal Gene Transfer: Methods and Protocols. New York, NY: Springer US; 2020. p. 265–83. pmid:31584169
- 40.
Garcillán-Barcia MP, Redondo-Salvo S, Vielva L, de la Cruz F. MOBscan: Automated annotation of MOB relaxases. In: de la Cruz F, editor. Horizontal Gene Transfer: Methods and Protocols. New York, NY: Springer US; 2020. p. 295–308.
- 41. Alcock BP, Huynh W, Chalil R, Smith KW, Raphenya Amogelang R, Wlodarski MA, et al. CARD 2023: expanded curation, support for machine learning, and resistome prediction at the Comprehensive Antibiotic Resistance Database. Nucleic Acids Res. 2023;51(D1):D690–D9. https://doi.org10.1093/nar/gkac920
- 42. Chen L, Yang J, Yu J, Yao Z, Sun L, Shen Y, et al. VFDB: a reference database for bacterial virulence factors. Nucleic Acids Res. 2005;33(Supplement 1):D325–8. pmid:15608208
- 43. Seemann T. ABRicate. 2020 [cited 1 May 2024]. Available from: https://github.com/tseemann/abricate.
- 44. Wishart DS, Han S, Saha S, Oler E, Peters H, Grant Jason R, et al. PHASTEST: faster than PHASTER, better than PHAST. Nucleic Acids Res. 2023;51(W1):W443–W50. pmid:37194694
- 45. Eren AM, Kiefl E, Shaiber A, Veseli I, Miller SE, Schechter MS, et al. Community-led, integrated, reproducible multi-omics with anvi’o. Nat Microbiol. 2021;6(1):3–6. pmid:33349678
- 46. Foley S L., Kaldhone P R., Ricke S C., Han J. Incompatibility group I1 (IncI1) plasmids: Their genetics, biology, and public health relevance. Microbiology and molecular biology reviews: MMBR. 2021;85(2):e00031–20. pmid:33910982
- 47. Freire Martín I, AbuOun M, Reichel R, La Ragione RM, Woodward MJ. Sequence analysis of a CTX-M-1 IncI1 plasmid found in Salmonella 4,5,12:i:−, Escherichia coli and Klebsiella pneumoniae on a UK pig farm. J Antimicrob Chemother. 2014;69(8):2098–101. https://doi.org10.1093/jac/dku098
- 48. Gulig PA, Curtiss R, 3rd. Plasmid-associated virulence of Salmonella typhimurium. Infection and immunity. 1987;55(12):2891–901. https://doi.org10.1128/iai.55.12.2891-2901.1987
- 49. Oluwadare M, Lee MD, Grim CJ, Lipp EK, Cheng Y, Maurer JJ. The role of the Salmonella spvB IncF plasmid and its resident entry exclusion gene traS on plasmid exclusion. Frontiers in microbiology. 2020;11:949. https://doi.org10.3389/fmicb.2020.00949
- 50. Silva C, Puente JL, Calva E. Salmonella virulence plasmid: pathogenesis and ecology. Pathog Dis. 2017;75(6). https://doi.org10.1093/femspd/ftx070
- 51. Chen Z, Kuang D, Xu X, González-Escalona N, Erickson DL, Brown E, et al. Genomic analyses of multidrug-resistant Salmonella Indiana, Typhimurium, and Enteritidis isolates using MinION and MiSeq sequencing technologies. PloS One. 2020;15(7):e0235641. https://doi.org10.1371/journal.pone.0235641
- 52. Gorski L, Huynh S, Cooper K, K., Parker C, T. Complete genomic sequences of two Salmonella enterica subsp. enterica serogroup C2 (O:6,8) strains from Central California. Genome Announc. 2017;5(46):e01234–17. https://doi.org10.1128/genomea.01234-17
- 53. Zayas CL, Claas K, Escalante-Semerena JC. The CbiB protein of Salmonella enterica is an integral membrane protein involved in the last step of the de novo corrin ring biosynthetic pathway. J Bacteriol. 2007;189(21):7697–708. https://doi.org10.1128/jb.01090-07
- 54. Pfeifer E, Rocha EPC. Phage-plasmids promote recombination and emergence of phages and plasmids. Nat Commun. 2024;15(1):1545. pmid:38378896
- 55. Kidgell C, Pickard D, Wain J, James K, Diem Nga LT, Diep TS, et al. Characterisation and distribution of a cryptic Salmonella typhi plasmid pHCM2. Plasmid. 2002;47(3):159–71. https://doi.org10.1016/s0147-619x(02)00013-6
- 56. Octavia S, Sara J, Lan R. Characterization of a large novel phage-like plasmid in Salmonella enterica serovar Typhimurium. FEMS microbiology letters. 2015;362(8):fnv044. https://doi.org10.1093/femsle/fnv044
- 57. Greig DR, Bird MT, Chattaway MA, Langridge GC, Waters EV, Ribeca P, et al. Characterization of a P1-bacteriophage-like plasmid (phage-plasmid) harbouring blaCTX-M-15 in Salmonella enterica serovar Typhi. Microb Genom. 2022;8(12):mgen000913. https://doi.org10.1099/mgen.0.000913
- 58. Leeper MM, Tolar BM, Griswold T, Vidyaprakash E, Hise KB, Williams GM, et al. Evaluation of whole and core genome multilocus sequence typing allele schemes for Salmonella enterica outbreak detection in a national surveillance network, PulseNet USA. Frontiers in microbiology. 2023;14:1254777. https://doi.org10.3389/fmicb.2023.1254777
- 59. Badrinarayanan A, Le TBK, Laub MT. Rapid pairing and resegregation of distant homologous loci enables double-strand break repair in bacteria. J Cell Biol. 2015;210(3):385–400. pmid:26240183
- 60. Köhler R, Kaganovitch E, Murray SM. High-throughput imaging and quantitative analysis uncovers the nature of plasmid positioning by ParABS. eLife. 2022;11:e78743. pmid:36374535
- 61. Midonet C, Barre F-X. Xer site-specific recombination: Promoting vertical and horizontal transmission of genetic information. Microbiol Spectr. 2014;2(6):MDNA3-0056-2014. pmid:26104463
- 62. Wood TL, Wood TK. The HigB/HigA toxin/antitoxin system of Pseudomonas aeruginosa influences the virulence factors pyochelin, pyocyanin, and biofilm formation. MicrobiologyOpen. 2016;5(3):499–511. https://doi.org10.1002/mbo3.346
- 63. Jurėnas D, Fraikin N, Goormaghtigh F, Van Melderen L. Biology and evolution of bacterial toxin-antitoxin systems. Nat Rev Microbiol. 2022;20(6):335–50. pmid:34975154
- 64. Kamruzzaman M, Wu AY, Iredell JR. Biological functions of type II toxin-antitoxin systems in bacteria. Microorganisms. 2021;9(6):1276. pmid:34208120
- 65. McMahon SA, Roberts GA, Johnson KA, Cooper LP, Liu H, White JH, et al. Extensive DNA mimicry by the ArdA anti-restriction protein and its role in the spread of antibiotic resistance. Nucleic Acids Res. 2009;37(15):4887–97. pmid:19506028
- 66. Sabrin A, Gioe BW, Gupta A, Grove A. An EmrB multidrug efflux pump in Burkholderia thailandensis with unexpected roles in antibiotic resistance. The Journal of biological chemistry. 2019;294(6):1891–903. https://doi.org10.1074/jbc.RA118.006638
- 67. Zhang H, Ma Y, Liu P, Li X. Multidrug resistance operon emrAB contributes for chromate and ampicillin co-resistance in a Staphylococcus strain isolated from refinery polluted river bank. SpringerPlus. 2016;5(1):1648. pmid:27722066
- 68. Crawford MA, Henard CA, Tapscott T, Porwollik S, McClelland M, Vázquez-Torres A. DksA-dependent transcriptional regulation in Salmonella experiencing nitrosative stress. Frontiers in microbiology. 2016;7:444. https://doi.org10.3389/fmicb.2016.00444
- 69. Roux N, Spagnolo J, de Bentzmann S. Neglected but amazingly diverse type IVb pili. Research in microbiology. 2012;163(9–10):659–73. pmid:23103334
- 70. Little JI, Singh PK, Zhao J, Dunn S, Matz H, Donnenberg MS. Type IV pili of Enterobacteriaceae species. EcoSal Plus. 2024:eesp00032023. https://doi.org10.1128/ecosalplus.esp-0003-2023
- 71. Zhang XL, Tsui IS, Yip CM, Fung AW, Wong DK, Dai X, et al. Salmonella enterica serovar Typhi uses type IVB pili to enter human intestinal epithelial cells. Infection and immunity. 2000;68(6):3067–73. https://doi.org10.1128/iai.68.6.3067-3073.2000
- 72. Marković KG, Grujović MŽ, Koraćević MG, Nikodijević DD, Milutinović MG, Semedo-Lemsaddek T, et al. Colicins and microcins produced by Enterobacteriaceae: Characterization, mode of action, and putative applications. Int J Environ Res Public Health. 2022;19(18):11825. https://doi.org10.3390/ijerph191811825
- 73. Baquero F, Lanza VF, Baquero M-R, del Campo R, Bravo-Vázquez DA. Microcins in Enterobacteriaceae: Peptide antimicrobials in the eco-active intestinal chemosphere. Frontiers in microbiology. 2019;10:2261. https://doi.org10.3389/fmicb.2019.02261
- 74. Vasicek EM, O’Neal L, Parsek MR, Fitch J, White P, Gunn JS. L-arabinose transport and metabolism in Salmonella influences biofilm formation. Front Cell Infect Microbiol. 2021;11:698146. https://doi.org10.3389/fcimb.2021.698146
- 75. Quistgaard EM, Löw C, Guettou F, Nordlund P. Understanding transport by the major facilitator superfamily (MFS): structures pave the way. Nat Rev Mol Cell Biol. 2016;17(2):123–32. pmid:26758938
- 76. Drew D, North RA, Nagarathinam K, Tanabe M. Structures and general transport mechanisms by the major facilitator superfamily (MFS). Chem Rev. 2021;121(9):5289–335. pmid:33886296
- 77. Bergman S, Andresen L, Kjellin J, Martinez Burgo Y, Geiser P, Baars S, et al. ProQ-dependent activation of Salmonella virulence genes mediated by post-transcriptional control of PhoP synthesis. mSphere. 2024;9(3):e00018–24. pmid:38411119
- 78. Dik DA, Marous DR, Fisher JF, Mobashery S. Lytic transglycosylases: concinnity in concision of the bacterial cell wall. Crit Rev Biochem Mol Biol. 2017;52(5):503–42. pmid:28644060
- 79. Kim H-S, Kim S-M, Lee H-J, Park S-J, Lee K-H. Expression of the cpdA gene, encoding a 3′,5′-cyclic AMP (cAMP) phosphodiesterase, is positively regulated by the cAMP-cAMP receptor protein complex. J Bacteriol. 2009;191(3):922–30. pmid:19028903
- 80. Ross K, Varani Alessandro M, Snesrud E, Huang H, Alvarenga Danillo O, Zhang J, et al. TnCentral: A prokaryotic transposable element database and web portal for transposon analysis. mBio. 2021;12(5):e0206021. pmid:34517763
- 81. Haniford David B, Ellis Michael J. Transposons Tn10 and Tn5. Microbiol Spectr. 2015;3(1):MDNA3-0002-2014. pmid:26104553
- 82. Garrido V, Arrieta-Gisasola A, Migura-García L, Laorden L, Grilló MJ. Multidrug resistance in Salmonella isolates of swine origin: mobile genetic elements and plasmids associated with cephalosporin resistance with potential transmission to humans. Appl Environ Microbiol 2024;90(5):e00264–24. https://doi.org10.1128/aem.00264-24
- 83. Manyi-Loh C, Mamphweli S, Meyer E, Okoh A. Antibiotic use in agriculture and its consequential resistance in environmental sources: Potential public health implications. Molecules. 2018;23(4):795. pmid:29601469
- 84. McMillan EA, Gupta SK, Williams LE, Jové T, Hiott LM, Woodley TA, et al. Antimicrobial resistance genes, cassettes, and plasmids present in Salmonella enterica associated with United States food animals. Frontiers in microbiology. 2019;10:832. https://doi.org10.3389/fmicb.2019.00832
- 85. Nekrasov SV, Agafonova OV, Belogurova NG, Delver EP, Belogurov AA. Plasmid-encoded antirestriction protein ArdA can discriminate between type I methyltransferase and complete restriction-modification system. Journal of molecular biology. 2007;365(2):284–97. pmid:17069852