Genetic relatedness and virulence potential of Salmonella Schwarzengrund strains with or without an IncFIB-IncFIC(FII) fusion plasmid isolated from food and clinical sources.

A total of 55 food and clinical S. Schwarzengrund isolates were assayed for plasmid content, among which an IncFIB-IncFIC(FII) fusion plasmid, conferring streptomycin resistance, was detected in 17 isolates. Among the 17 isolates, 9 were food isolates primarily collected from poultry meat, and 8 clinical isolates collected from stool, urine, and gallbladder. SNP-based phylogenetic analyses showed that the isolates carrying the fusion plasmid formed a subclade indicating the plasmid was acquired and is now maintained by the lineage. Phylogenetic analysis of the plasmid suggested it is derived from avian pathogenic plasmids and might confer an adaptive advantage to the S. Schwarzengrund isolates within birds. IncFIB-IncFIC(FII) fusion plasmids from all food and three clinical isolates were self-conjugative and successfully transferred into E. coli J53 by conjugation. Food and clinical isolates had similar virulome profiles and were able to invade human Caco-2 cells. However, the IncFIB-IncFIC(FII) plasmid did not significantly add to their invasion and persistence potential in human Caco-2 cells.

Complete genome sequences of 17 Salmonella enterica serovar Schwarzengrund isolates carrying an IncFIB-IncFIC (FII) fusion plasmid.

Seventeen Salmonella enterica serovar Schwarzengrund isolates from chicken (n = 9) and clinical samples including stool (n = 6), urine (n = 1), and gallbladder (n = 1) were sequenced and found to carry an IncFIB-IncFIC (FII) fusion plasmid of approximately 145 Kb. This information provides reference genomic data for comparative studies of S. Schwarzengrund pathogenicity and plasmid genetics.

Development of an antimicrobial resistance plasmid transfer gene database for enteric bacteria.

Introduction: Type IV secretion systems (T4SSs) are integral parts of the conjugation process in enteric bacteria. These secretion systems are encoded within the transfer (tra) regions of plasmids, including those that harbor antimicrobial resistance (AMR) genes. The conjugal transfer of resistance plasmids can lead to the dissemination of AMR among bacterial populations. Methods: To facilitate the analyses of the conjugation-associated genes, transfer related genes associated with key groups of AMR plasmids were identified, extracted from GenBank and used to generate a plasmid transfer gene dataset that is part of the Virulence and Plasmid Transfer Factor Database at FDA, serving as the foundation for computational tools for the comparison of the conjugal transfer genes. To assess the genetic feature of the transfer gene database, genes/proteins of the same name (e.g., traI/TraI) or predicted function (VirD4 ATPase homologs) were compared across the different plasmid types to assess sequence diversity. Two analyses tools, the Plasmid Transfer Factor Profile Assessment and Plasmid Transfer Factor Comparison tools, were developed to evaluate the transfer genes located on plasmids and to facilitate the comparison of plasmids from multiple sequence files. To assess the database and associated tools, plasmid, and whole genome sequencing (WGS) data were extracted from GenBank and previous WGS experiments in our lab and assessed using the analysis tools. Results: Overall, the plasmid transfer database and associated tools proved to be very useful for evaluating the different plasmid types, their association with T4SSs, and increased our understanding how conjugative plasmids contribute to the dissemination of AMR genes.

Microbial Genetics and Clonal Dissemination of Salmonella enterica Serotype Javiana Isolated from Human Populations in Arkansas, USA

Salmonella is estimated to cause over a million infections and ~400 deaths annually in the U.S. Salmonella enterica serotype Javiana strains (n = 409) that predominantly originated from the State of Arkansas over a six-year period (2003 to 2008) were studied. This period coincided with a rapid rise in the incidence of S. Javiana infections in the U.S. Children under the age of 10 displayed the highest prevalence of S. Javiana infections, regardless of sex or year of detection. Antimicrobial susceptibility to 15 different antimicrobials was assessed and 92% (n = 375) were resistant to at least one of the antimicrobials. Approximately 89% of the isolates were resistant to sulfisoxazole alone and 3% (n = 11) were resistant to different antimicrobials, including gentamicin, ciprofloxacin or ceftiofur. The pulsed-field gel electrophoresis (PFGE) analyses assessed the genotypic diversity and distribution of S. Javiana strains using XbaI restriction. Nine major clusters were identified and isolates from each group were digested with the restriction enzyme AvrII. Isolates with identical profiles of XbaI and AvrII were found to be disseminated in human populations. These distinct "types" of S. Javiana were persistent in human populations for multiple years. A subset of isolates (n = 19) with unique resistance phenotypes underwent plasmid and incompatibility (Inc) type analyses and the isolates resistant to more than one antimicrobial harbored multiple plasmids (<3 to 165 kb). Furthermore, these strains possessed 14 virulence genes, including pagC, cdtB, and iroN. The whole genome sequences (WGS) of 18 isolates that mostly originated from Arkansas from 2003 to 2011 were compared with isolates collected from different areas in the U.S. in 1999, indicating the perseverance of S. Javiana in disseminating antimicrobial resistance and virulence genes.

Antimicrobial Resistance and Increased Virulence of Salmonella.

This special issue of Microorganisms highlights the importance of antimicrobial resistance (AMR) and increased virulence of Salmonella with multiple research papers [...].

In silico analyses of diversity and dissemination of antimicrobial resistance genes and mobile genetics elements, for plasmids of enteric pathogens.

Introduction: The antimicrobial resistance (AMR) mobilome plays a key role in the dissemination of resistance genes encoded by mobile genetics elements (MGEs) including plasmids, transposons (Tns), and insertion sequences (ISs). These MGEs contribute to the dissemination of multidrug resistance (MDR) in enteric bacterial pathogens which have been considered as a global public health risk. Methods: To further understand the diversity and distribution of AMR genes and MGEs across different plasmid types, we utilized multiple sequence-based computational approaches to evaluate AMR-associated plasmid genetics. A collection of 1,309 complete plasmid sequences from Gammaproteobacterial species, including 100 plasmids from each of the following 14 incompatibility (Inc) types: A/C, BO, FIA, FIB, FIC, FIIA, HI1, HI2, I1, K, M, N, P except W, where only 9 sequences were available, was extracted from the National Center for Biotechnology Information (NCBI) GenBank database using BLAST tools. The extracted FASTA files were analyzed using the AMRFinderPlus web-based tools to detect antimicrobial, disinfectant, biocide, and heavy metal resistance genes and ISFinder to identify IS/Tn MGEs within the plasmid sequences. Results and Discussion: In silico prediction based on plasmid replicon types showed that the resistance genes were diverse among plasmids, yet multiple genes were widely distributed across the plasmids from enteric bacterial species. These findings provide insights into the diversity of resistance genes and that MGEs mediate potential transmission of these genes across multiple plasmid replicon types. This notion was supported by the observation that many IS/Tn MGEs and resistance genes known to be associated with them were common across multiple different plasmid types. Our results provide critical insights about how the diverse population of resistance genes that are carried by the different plasmid types can allow for the dissemination of AMR across enteric bacteria. The results also highlight the value of computational-based approaches and in silico analyses for the assessment of AMR and MGEs, which are important elements of molecular epidemiology and public health outcomes.

Expression of Genes Located on the Incompatibility Group FIB Plasmids at Transcription and Protein Levels in Iron-Modified Growth Conditions.

Salmonella enterica strains often harbor plasmids representing several incompatibility groups (Inc) including IncFIB, which have been previously associated with carrying antimicrobial resistance and virulence associated genes. To better understand the distribution of virulence genes on IncFIB plasmids, we analyzed 37 complete whole genome and plasmid sequences of different S. enterica isolates from multiple serovars. Many of the sequences analyzed carried multiple virulence-associated genes, including those associated with iron acquisition systems; thus we aimed to determine how iron-rich (IR) and various iron-depleted (ID) conditions affected the transcription of iron acquisition and virulence genes including sitA, iutA, iucA, and enolase at different time intervals. sitA, iutA, and enolase from S. enterica that were grown in Luria-Bertani broth (LB) ID (LBID) conditions were substantially upregulated when compared to LBIR conditions. For both S. enterica strains that were grown at various LBID conditions, addition of 200 µM bipyridyl in the growth medium yielded the highest transcription for all four genes, followed by the 100 µM concentration. An antibody using a peptide targeting aerobactin receptor gene iutA encoded by IncFIB was generated and used to examine the protein expression in the wild-type, recipient, and transconjugant strain in LB, LBID, and LBIR growth conditions using Western blot analyses. A 70 KDa protein band was detected in the wild-type and transconjugant that carried the IncFIB plasmid, while this band was not detected in the recipient strain that lacked this plasmid.

Genotypic and Phenotypic Characterization of Incompatibility Group FIB Positive Salmonella enterica Serovar Typhimurium Isolates from Food Animal Sources.

Salmonella enterica is one of the most common bacterial foodborne pathogens in the United States, causing illnesses that range from self-limiting gastroenteritis to more severe, life threatening invasive disease. Many Salmonella strains contain plasmids that carry virulence, antimicrobial resistance, and/or transfer genes which allow them to adapt to diverse environments, and these can include incompatibility group (Inc) FIB plasmids. This study was undertaken to evaluate the genomic and phenotypic characteristics of IncFIB-positive Salmonella enterica serovar Typhimurium isolates from food animal sources, to identify their plasmid content, assess antimicrobial resistance and virulence properties, and compare their genotypic isolates with more recently isolated S. Typhimurium isolates from food animal sources. Methods: We identified 71 S. Typhimurium isolates that carried IncFIB plasmids. These isolates were subjected to whole genome sequencing and evaluated for bacteriocin production, antimicrobial susceptibility, the ability to transfer resistance plasmids, and a subset was evaluated for their ability to invade and persist in intestinal human epithelial cells. Results: Approximately 30% of isolates (n = 21) displayed bacteriocin inhibition of Escherichia coli strain J53. Bioinformatic analyses using PlasmidFinder software confirmed that all isolates contained IncFIB plasmids along with multiple other plasmid replicon types. Comparative analyses showed that all strains carried multiple antimicrobial resistance genes and virulence factors including iron acquisition genes, such as iucABCD (75%), iutA (94%), sitABCD (76%) and sitAB (100%). In 17 cases (71%), IncFIB plasmids, along with other plasmid replicon types, were able to conjugally transfer antimicrobial resistance and virulence genes to the susceptible recipient strain. For ten strains, persistence cell counts (27%) were noted to be significantly higher than invasion bacterial cell counts. When the genome sequences of the study isolates collected from 1998-2003 were compared to those published from subsequent years (2005-2018), overlapping genotypes were found, indicating the perseverance of IncFIB positive strains in food animal populations. This study confirms that IncFIB plasmids can play a potential role in disseminating antimicrobial resistance and virulence genes amongst bacteria from several food animal species.

Draft Genome Sequences of Two Campylobacter jejuni Strains That Show Significantly Different Colonization Potentials in Chickens.

Here, we report the draft genome sequences of robust (A74/C_24-3) and poor (A74/O_2-2) chicken-colonizing Campylobacter jejuni isolates. Whole-genome sequence analyses of these isolates will be helpful in facilitating further studies to identify genetic factors used in chicken colonization.

Whole-Genome Sequences of 66 Incompatibility Group FIB Plasmid-Carrying Salmonella enterica Serovar Typhimurium Isolates from Food Animal Sources.

Sixty-six Salmonella enterica serovar Typhimurium isolates carrying incompatibility group FIB (IncFIB) plasmids were sequenced to further characterize the IncFIB plasmid-encoded factors associated with virulence and antimicrobial resistance genes. In addition to the IncFIB plasmid, many of these isolates harbored additional plasmids encoding virulence and antimicrobial resistance genes.

Evaluation of Incompatibility Group I1 (IncI1) Plasmid-Containing Salmonella enterica and Assessment of the Plasmids in Bacteriocin Production and Biofilm Development.

Mobile genetic elements, such as plasmids, can potentially increase the ability of bacteria to infect and persist in vertebrate host cells. IncI1 plasmids are widely distributed in Salmonella from food animal sources and associated with clinically important strains. These plasmids often encode antimicrobial resistance; however, little is known about their impact on the virulence of Salmonella strains. To assess the potential impact of the plasmids on virulence, 43 IncI1-positive Salmonella isolates from human and animal sources were subjected to whole genome sequence (WGS) analyses and evaluated for their abilities to invade and persist for 48 h in Caco-2 human intestinal epithelial cells, form biofilms and encode bacteriocins. Draft WGS data were submitted to predict the presence of virulence and antimicrobial resistance genes, plasmid replicon types present, conduct plasmid multilocus sequence typing (pMLST), and core genome MLST (cgMLST) in the isolates. Caco-2 cells were infected with Salmonella strains and incubated for both one and 48 h for the invasion and persistence assays, respectively. Additionally, Salmonella isolates and IncI1 plasmid carrying transconjugants (n = 12) generated in Escherichia coli were assessed for their ability to produce biofilms and bacteriocin inhibition of growth of other bacteria. All Salmonella isolates infected Caco-2 cells and persisted in the cells at 48 hrs. Persistent cell counts were observed to be significantly higher than invasion assay cell counts in 26% of the isolates. Among the IncI1 plasmids, there were 18 pMLST types. Nearly 35% (n = 15) of Salmonella isolates produced biofilms; however, none of the IncI1-positive transconjugants produced increased biofilms compared to the recipient. Approximately 65% (n = 28) of isolates and 67% (n = 8) of IncI1-positive transconjugants were able to inhibit growth of at least one E. coli strain; however, none inhibited the growth of strains from species other than E. coli. The study characterized IncI1 positive Salmonella isolates and provided evidence about the potential contributions of IncI1 plasmids virulence phenotypes and areas where they do not. These findings should allow for more focused efforts to assess the impact of plasmids on bacterial pathophysiology and human health.

Whole-Genome Sequences of 35 Incompatibility Group I1 Plasmid-Carrying Salmonella enterica Isolates from Food Animal and Clinical Sources.

We sequenced 35 Salmonella enterica isolates carrying incompatibility group I1 (IncI1) plasmids from different serotypes to study their genotypic characteristics. The isolates originated from food animals (n = 32) and human patients (n = 3). All isolates carried IncI1 plasmids, and many had additional plasmids detected along with virulence and antimicrobial resistance genes.

Global transcriptomic analyses of Salmonella enterica in Iron-depleted and Iron-rich growth conditions.

BACKGROUND: Salmonella enterica possess several iron acquisition systems, encoded on the chromosome and plasmids. Recently, we demonstrated that incompatibility group (Inc) FIB plasmid-encoded iron acquisition systems (Sit and aerobactin) likely play an important role in persistence of Salmonella in human intestinal epithelial cells (Caco-2). In this study, we sought to determine global transcriptome analyses of S. enterica in iron-rich (IR) and iron-depleted (ID) growth conditions. RESULTS: The number of differentially-expressed genes were substantially higher for recipient (SE819) (n = 966) and transconjugant (TC) (n = 945) compared to the wild type (WT) (SE163A) (n = 110) strain in ID as compared to IR growth conditions. Several virulence-associated factors including T3SS, flagellin, cold-shock protein (cspE), and regulatory genes were upregulated in TC in ID compared to IR conditions. Whereas, IS1 and acrR/tetR transposases located on the IncFIB plasmid, ferritin and several regulatory genes were downregulated in TC in ID conditions. Enterobactin transporter (entS), iron ABC transporter (fepCD), colicin transporter, IncFIB-encoded enolase, cyclic di-GMP regulator (cdgR) and other regulatory genes of the WT strain were upregulated in ID compared to IR conditions. Conversely, ferritin, ferrous iron transport protein A (feoA), IncFIB-encoded IS1 and acrR/tetR transposases and ArtA toxin of WT were downregulated in ID conditions. SDS-PAGE coupled with LC-MS/MS analyses revealed that siderophore receptor proteins such as chromosomally-encoded IroN and, IncFIB-encoded IutA were upregulated in WT and TC in ID growth conditions. Both chromosome and IncFIB plasmid-encoded SitA was overexpressed in WT, but not in TC or recipient in ID conditions. Increased expression of flagellin was detected in recipient and TC, but not in WT in ID conditions. CONCLUSION: Iron concentrations in growth media influenced differential gene expressions both at transcriptional and translational levels, including genes encoded on the IncFIB plasmid. Limited iron availability within the host may promote pathogenic Salmonella to differentially express subsets of genes encoded by chromosome and/or plasmids, facilitating establishment of successful infection.

Protocols of Conjugative Plasmid Transfer in Salmonella: Plate, Broth, and Filter Mating Approaches.

Bacterial conjugation is a natural process that allows for horizontal transmission of DNA from one bacterium to another. Several plasmids carry transposons that encode multiple antimicrobial and metal resistance genes. Conjugative plasmid transfer requires intimate cell-to-cell contacts between the donor and the recipient. Self-conjugative plasmids harbor tra genes which facilitate plasmid transfer from donor to recipient bacterial strain. Here we describe different methods of conjugative plasmid transfers via conjugation.

Draft Genome Sequences of 27 Salmonella enterica Serovar Schwarzengrund Isolates from Clinical Sources.

Twenty-seven Salmonella enterica serovar Schwarzengrund isolates from clinical sources were sequenced as part of a larger study to examine phenotypic and genotypic characteristics. The majority of the sequenced strains were isolated from human stool (n = 20) followed by urine (n = 3) and blood (n = 2). Four isolate sequences contained plasmids of known incompatibility groups.

Draft Genome Sequences of Salmonella enterica Serovar Enteritidis and Kentucky Isolates from Retail Poultry Sources.

The draft genome sequences of four Salmonella enterica serovar Enteritidis and Kentucky isolates were evaluated for biofilm formation and antibiotic resistance. The Salmonella serovar Kentucky strains CFS84 and CFS85 and Salmonella serovar Enteritidis strains CFS86 and CFS87 were isolated from retail poultry sources in Arkansas.

Draft Genome Sequences of Ciprofloxacin-Resistant Salmonella enterica Strains with Multiple-Antibiotic Resistance, Isolated from Imported Foods.

We report here the draft genome sequences of 15 ciprofloxacin-resistant Salmonella enterica strains with resistance to multiple other antibiotics, including aminoglycosides, ß-lactams, sulfonamides, tetracycline, and trimethoprim, isolated from different imported foods. Three strains (NCTR75, NCTR281, and NCTR350) showed a high level of ciprofloxacin resistance compared to that of the other isolates. The whole-genome sequencing data provide a better understanding of the antibiotic resistance mechanisms and virulence properties of these isolates.

Draft Genome Sequences of Salmonella enterica Isolates Containing Incompatibility Group I1 Plasmids from Swine, Poultry, and Human Sources.

The draft genome sequences of eight Salmonella enterica isolates from various sources were evaluated for the influence of incompatibility group I1 (IncI1) plasmids on virulence. Strains SE142, SE143, SE144, and SE146 originated from swine, SE36N and SE89N from poultry-related sources, and SE991 and SE1148 from human patients.

Comparative genomic analysis and characterization of incompatibility group FIB plasmid encoded virulence factors of Salmonella enterica isolated from food sources.

BACKGROUND: The degree to which the chromosomal mediated iron acquisition system contributes to virulence of many bacterial pathogens is well defined. However, the functional roles of plasmid encoded iron acquisition systems, specifically Sit and aerobactin, have yet to be determined for Salmonella spp. In a recent study, Salmonella enterica strains isolated from different food sources were sequenced on the Illumina MiSeq platform and found to harbor the incompatibility group (Inc) FIB plasmid. In this study, we examined sequence diversity and the contribution of factors encoded on the IncFIB plasmid to the virulence of S. enterica. RESULTS: Whole genome sequences of seven S. enterica isolates were compared to genomes of serovars of S. enterica isolated from food, animal, and human sources. SeqSero analysis predicted that six strains were serovar Typhimurium and one was Heidelberg. Among the S. Typhimurium strains, single nucleotide polymorphism (SNP)-based phylogenetic analyses revealed that five of the isolates clustered as a single monophyletic S. Typhimurium subclade, while one of the other strains branched with S. Typhimurium from a bovine source. DNA sequence based phylogenetic diversity analyses showed that the IncFIB plasmid-encoded Sit and aerobactin iron acquisition systems are conserved among bacterial species including S. enterica. The IncFIB plasmid was transferred to an IncFIB plasmid deficient strain of S. enterica by conjugation. The transconjugant SE819::IncFIB persisted in human intestinal epithelial (Caco-2) cells at a higher rate than the recipient SE819. Genes of the Sit and aerobactin operons in the IncFIB plasmid were differentially expressed in iron-rich and iron-depleted growth media. CONCLUSIONS: Minimal sequence diversity was detected in the Sit and aerobactin operons in the IncFIB plasmids present among different bacterial species, including foodborne Salmonella strains. IncFIB plasmid encoded factors play a role during infection under low-iron conditions in host cells.

Draft Genome Sequences of Four Salmonella enterica Strains Isolated from Turkey-Associated Sources.

We report the draft genomes of four Salmonella enterica isolates evaluated for the contribution of plasmids to virulence. Strains SE163A, SE696A, and SE710A carry plasmids demonstrated to facilitate plasmid-associated virulence, while SE819 is less virulent and has been used as a recipient for conjugation experiments to assess plasmid-encoded virulence mechanisms.