Recently, tomatoes have been implicated as a primary vehicle in food-borne outbreaks of Salmonella enterica serovar Newport and other Salmonella serovars. Long-term intervention measures to reduce Salmonella prevalence on tomatoes remain elusive for growing and postharvest environments. A naturally occurring bacterium identified by 16S rRNA gene sequencing as Paenibacillus alvei was isolated epiphytically from plants native to the Virginia Eastern Shore tomato-growing region. After initial antimicrobial activity screening against Salmonella and 10 other bacterial pathogens associated with the human food supply, strain TS-15 was further used to challenge an attenuated strain of S. Newport on inoculated fruits, leaves, and blossoms of tomato plants in an insect-screened high tunnel with a split-plot design. Survival of Salmonella after inoculation was measured for groups with and those without the antagonist at days 0, 1, 2, and 3 and either day 5 for blossoms or day 6 for fruits and leaves. Strain TS-15 exhibited broad-range antimicrobial activity against both major food-borne pathogens and major bacterial phytopathogens of tomato. After P. alvei strain TS-15 was applied onto the fruits, leaves, and blossoms of tomato plants, the concentration of S. Newport declined significantly (P ≤ 0.05) compared with controls. Astonishingly, >90% of the plants had no detectable levels of Salmonella by day 5 for blossoms. The naturally occurring antagonist strain TS-15 is highly effective in reducing the carriage of Salmonella Newport on whole tomato plants. The application of P. alvei strain TS-15 is a promising approach for reducing the risk of Salmonella contamination during tomato production.
Here we report the genome sequence of a Clostridium botulinum strain IBCA10-7060 producing botulinum neurotoxin serotype B and a new toxin serotype. Multilocus sequence typing analysis revealed that this strain belongs to a new sequence type, and whole-genome single nucleotide polymorphism analysis showed that this strain clustered with strains in lineage 2 from group I.
Phage typing has been used for the epidemiological surveillance of Salmonella enterica serovar Enteritidis for over 2 decades. However, knowledge of the genetic and evolutionary relationships between phage types is very limited, making differences difficult to interpret. Here, single nucleotide polymorphisms (SNPs) identified from whole-genome comparisons were used to determine the relationships between some S. Enteritidis phage types (PTs) commonly associated with food-borne outbreaks in the United States. Emphasis was placed on the predominant phage types PT8, PT13a, and PT13 in North America. With >89,400 bp surveyed across 98 S. Enteritidis isolates representing 14 distinct phage types, 55 informative SNPs were discovered within 23 chromosomally anchored loci. To maximize the discriminatory and evolutionary partitioning of these highly homogeneous strains, sequences comprising informative SNPs were concatenated into a single combined data matrix and subjected to phylogenetic analysis. The resultant phylogeny allocated most S. Enteritidis isolates into two distinct clades (clades I and II) and four subclades. Synapomorphic (shared and derived) sets of SNPs capable of distinguishing individual clades/subclades were identified. However, individual phage types appeared to be evolutionarily disjunct when mapped to this phylogeny, suggesting that phage typing may not be valid for making phylogenetic inferences. Furthermore, the set of SNPs identified here represents useful genetic markers for strain differentiation of more clonal S. Enteritidis strains and provides core genotypic markers for future development of a SNP typing scheme with S. Enteritidis.
Salmonella enterica subsp. enterica serovar Cubana (Salmonella serovar Cubana) is associated with human and animal disease. Here, we used third-generation, single-molecule, real-time DNA sequencing to determine the first complete genome sequence of Salmonella serovar Cubana CFSAN002050, which was isolated from fresh alfalfa sprouts during a multistate outbreak in 2012.
Rapid molecular typing methods are important tools in surveillance and outbreak investigations of human Salmonella infections. Here we described the development of a three-genes PCR-RFLP typing method for the differentiation of Salmonella species, subspecies and serovars using the Agilent 2100 Bioanalyzer. The fliC, gnd, and mutS genes were PCR-amplified in 160 Salmonella strains representing the two Salmonella species, six subspecies, and 41 different serovars of S. enterica subspecies enterica. PCR products were individually cut with two different restriction enzymes and the resulting 930 restriction patterns were collected using the Agilent 2100 Bioanalyzer followed by cluster analysis. Both species of Salmonella were differentiated by conventional PCR. All of S. bongori tested were gnd PCR negative due to a mismatch at the 3′-end in one the PCR primers. Salmonella subspecies were differentiated into third-teen homogeneous groups representing each of the six subspecies by cluster analysis of restriction patterns generated from the mutS gene cut with AciI. S. enterica subspecies enterica serovars were further differentiated by the combination of the three target genes and five out the six sets of restriction patterns with a discriminatory power of 0.9725 by cluster analysis. The combined RFLP results of five sets of restriction patterns allowed us to assign each of the 160 strains to one of 128 restriction types. During inoculation studies we were able to identify S. Saintpaul and Typhimurium from 24 h pre-enrichment samples using the described method. The use of fliC, gnd, and mutS PCR-RFLP with the Agilent 2100 Bioanalyzer can provide an accessible and automated alternative method for differentiation of Salmonella pathogens.
Salmonella enterica; Bioanalyzer; PCR-RFLP; restriction type; reference collection
Vibrio parahaemolyticus is the leading cause of food-borne illnesses associated with the consumption of raw shellfish worldwide. Here, we report 45 draft genomes of V. parahaemolyticus. Thirty-five of them are strains that were isolated from clinical cases in the state of Maryland from 2010 to 2013. The remaining 10 strains were historical isolates, isolated mostly from the West Coast of the United States during the period of 1988 to 2004. The availability of these genomes will allow for future phylogenetic analyses with other V. parahaemolyticus strains.
For Salmonella enterica serovar Enteritidis, 85% of isolates can be classified into 5 pulsed-field gel electrophoresis (PFGE) types. However, PFGE has limited discriminatory power for outbreak detection. Although whole-genome sequencing has been found to improve discrimination of outbreak clusters, whether this procedure can be used in real-time in a public health laboratory is not known. Therefore, we conducted a retrospective and prospective analysis. The retrospective study investigated isolates from 1 confirmed outbreak. Additional cases could be attributed to the outbreak strain on the basis of whole-genome data. The prospective study included 58 isolates obtained in 2012, including isolates from 1 epidemiologically defined outbreak. Whole-genome sequencing identified additional isolates that could be attributed to the outbreak, but which differed from the outbreak-associated PFGE type. Additional putative outbreak clusters were detected in the retrospective and prospective analyses. This study demonstrates the practicality of implementing this approach for outbreak surveillance in a state public health laboratory.
Salmonella enterica serovar Enteritidis; bacteria; high-throughput nucleotide sequencing; whole-genome sequencing; pulsed-field gel electrophoresis; infectious disease outbreaks; public health laboratory surveillance
Next-generation sequencing is being evaluated for use with food-borne illness investigations, especially when the outbreak strains produce patterns that cannot be discriminated from non-outbreak strains using conventional procedures. Here we report complete genome assemblies of two Salmonella enterica serovar Heidelberg strains with a common pulsed-field gel electrophoresis pattern isolated during an outbreak investigation.
Evolutionary studies of clustered regularly interspaced short palindromic repeats (CRISPRs) and their associated (cas) genes can provide insights into host-pathogen co-evolutionary dynamics and the frequency at which different genomic events (e.g., horizontal vs. vertical transmission) occur. Within this study, we used whole genome sequence (WGS) data to determine the evolutionary history and genetic diversity of CRISPR loci and cas genes among a diverse set of 427 Salmonella enterica ssp. enterica isolates representing 64 different serovars. We also evaluated the performance of CRISPR loci for typing when compared to whole genome and multilocus sequence typing (MLST) approaches. We found that there was high diversity in array length within both CRISPR1 (median = 22; min = 3; max = 79) and CRISPR2 (median = 27; min = 2; max = 221). There was also much diversity within serovars (e.g., arrays differed by as many as 50 repeat-spacer units among Salmonella ser. Senftenberg isolates). Interestingly, we found that there are two general cas gene profiles that do not track phylogenetic relationships, which suggests that non-vertical transmission events have occurred frequently throughout the evolutionary history of the sampled isolates. There is also considerable variation among the ranges of pairwise distances estimated within each cas gene, which may be indicative of the strength of natural selection acting on those genes. We developed a novel clustering approach based on CRISPR spacer content, but found that typing based on CRISPRs was less accurate than the MLST-based alternative; typing based on WGS data was the most accurate. Notwithstanding cost and accessibility, we anticipate that draft genome sequencing, due to its greater discriminatory power, will eventually become routine for traceback investigations.
Salmonella; Horizontal gene transfer; Evolution; CRISPR; Outbreak; Phylogeny; Whole genome sequencing; Typing
bacteria; pandemic; Vibrio parahaemolyticus; Maryland; USA; United States; cross contamination; food preparation; traceback; PFGE pattern; pulsed-field gel electrophoresis pattern; seafood; epidemiology; gastroenteritis
Salmonella enterica subsp. enterica serovar Typhimurium is a leading cause of salmonellosis. Here, we report a closed genome sequence, including sequences of 3 plasmids, of Salmonella serovar Typhimurium var. 5− CFSAN001921 (National Antimicrobial Resistance Monitoring System [NARMS] strain ID N30688), which was isolated from chicken breast meat and shows resistance to 10 different antimicrobials. Whole-genome and plasmid sequence analyses of this isolate will help enhance our understanding of this pathogenic multidrug-resistant serovar.
Comparative methods for analyzing whole genome sequence (WGS) data enable us to assess the genetic information available for reconstructing the evolutionary history of pathogens. We used the comparative approach to determine diagnostic genes for Salmonella enterica subspecies I. S. enterica subsp. I strains are known to infect warm-blooded organisms regularly while its close relatives tend to infect only cold-blooded organisms. We found 71 genes gained by the common ancestor of Salmonella enterica subspecies I and not subsequently lost by any member of this subspecies sequenced to date. These genes included many putative functional phenotypes. Twenty-seven of these genes are found only in Salmonella enterica subspecies I; we designed primers to test these genes for use as diagnostic sequence targets and data mined the NCBI Sequence Read Archive (SRA) database for draft genomes which carried these genes. We found that the sequence specificity and variability of these amplicons can be used to detect and discriminate among 317 different serovars and strains of Salmonella enterica subspecies I.
The enteric pathogen Salmonella enterica is one of the leading causes of foodborne illness in the world. The species is extremely diverse, containing more than 2,500 named serovars that are designated for their unique antigen characters and pathogenicity profiles—some are known to be virulent pathogens, while others are not. Questions regarding the evolution of pathogenicity, significance of antigen characters, diversity of clustered regularly interspaced short palindromic repeat (CRISPR) loci, among others, will remain elusive until a strong evolutionary framework is established. We present the first large-scale S. enterica subsp. enterica phylogeny inferred from a new reference-free k-mer approach of gathering single nucleotide polymorphisms (SNPs) from whole genomes. The phylogeny of 156 isolates representing 78 serovars (102 were newly sequenced) reveals two major lineages, each with many strongly supported sublineages. One of these lineages is the S. Typhi group; well nested within the phylogeny. Lineage-through-time analyses suggest there have been two instances of accelerated rates of diversification within the subspecies. We also found that antigen characters and CRISPR loci reveal different evolutionary patterns than that of the phylogeny, suggesting that a horizontal gene transfer or possibly a shared environmental acquisition might have influenced the present character distribution. Our study also shows the ability to extract reference-free SNPs from a large set of genomes and then to use these SNPs for phylogenetic reconstruction. This automated, annotation-free approach is an important step forward for bacterial disease tracking and in efficiently elucidating the evolutionary history of highly clonal organisms.
H antigens; serovar; O antigens; CRISPR; lineage-through-time plot; comparative method
We sequenced the genomes of two strains of O104:H21 enterohemorrhagic Escherichia coli (EHEC) isolated during an outbreak of hemorrhagic colitis in Montana in 1994. These strains carried a plasmid that contains several virulence genes not present in pO157. The genome sequences will improve phylogenetic analysis of other non-O157 E. coli strains in the future.
The Salmonella enterica strains that are representatives of the S. enterica serovar Typhimurium complex in reference collection A (SARA) are closely related but exhibit differences in antibiotic resistance, which could have public health consequences. To better understand the mechanisms behind these resistances, we sequenced the genomes of two multidrug-resistant strains: SARA64 (Muenchen) and SARA33 (Heidelberg).
The consumption of fresh tomatoes has been linked to numerous food-borne outbreaks involving various serovars of Salmonella enterica. Recent advances in our understanding of plant-microbe interactions have shown that human enteric pathogenic bacteria, including S. enterica, are adapted to survive in the plant environment. In this study, tomato plants (Solanum lycopersicum cv. Micro-Tom) grown in sandy loam soil from Virginia's eastern shore (VES) were inoculated with S. enterica serovars to evaluate plausible internalization routes and to determine if there is any niche fitness for certain serovars. Both infested soil and contaminated blossoms can lead to low internal levels of fruit contamination with Salmonella. Salmonella serovars demonstrated a great ability to survive in environments under tomato cultivation, not only in soil but also on different parts of the tomato plant. Of the five serovars investigated, Salmonella enterica serovars Newport and Javiana were dominant in sandy loam soil, while Salmonella enterica serovars Montevideo and Newport were more prevalent on leaves and blossoms. It was also observed that Salmonella enterica serovar Typhimurium had a poor rate of survival in all the plant parts examined here, suggesting that postharvest contamination routes are more likely in S. Typhimurium contamination of tomato fruit. Conversely, S. Newport was the most prevalent serovar recovered in both the tomato rhizosphere and phyllosphere. Plants that were recently transplanted (within 3 days) had an increase in observable internalized bacteria, suggesting that plants were more susceptible to internalization right after transplant. These findings suggest that the particular Salmonella serovar and the growth stage of the plant were important factors for internalization through the root system.
To improve pulsed-field gel electrophoresis–based strain discrimination of 76 Salmonella Enteritidis strains, we evaluated 6 macro-restriction endonucleases, separately and in various combinations. One 3-enzyme subset, SfiI/PacI/NotI, was highly discriminatory. Five different indices, including the Simpson diversity index, supported this 3-enzyme combination for improved differentiation of S. Enteritidis.
Salmonella Enteritidis; subtyping; differentiation; pulsed-field gel electrophoresis; molecular epidemiology; clone; restriction endonuclease; genetic diversity; dispatch
Here, we report draft genomes of Paenibacillus alvei strains A6-6i and TS-15, which were isolated, respectively, from plant material and soil in the Virginia Eastern Shore (VES) tomato growing area. An array of genes related to antimicrobial biosynthetic pathways have been identified with whole-genome analyses of these strains.
Salmonella enterica subsp. enterica serovar Enteritidis is a common food-borne pathogen, often associated with shell eggs and poultry. Here, we report draft genomes of 21 S. Enteritidis strains associated with or related to the U.S.-wide 2010 shell egg recall. Eleven of these genomes were from environmental isolates associated with the egg outbreak, and 10 were reference isolates from previous years, unrelated to the outbreak. The whole-genome sequence data for these 21 human pathogen strains are being released in conjunction with the newly formed 100K Genome Project.
We report a closed genome of Salmonella enterica subsp. enterica serovar Javiana (S. Javiana). This serotype is a common food-borne pathogen and is often associated with fresh-cut produce. Complete (finished) genome assemblies will support pilot studies testing the utility of next-generation sequencing (NGS) technologies in public health laboratories.
The standard procedure for definitive detection of BoNT-producing Clostridia is a culture method combined with neurotoxin detection using a standard mouse bioassay (MBA). The mouse bioassay is highly sensitive and specific, but it is expensive and time-consuming, and there are ethical concerns due to use of laboratory animals. Cell-based assays provide an alternative to the MBA in screening for BoNT-producing Clostridia. Here, we describe a cell-based assay utilizing a fluorescence reporter construct expressed in a neuronal cell model to study toxin activity in situ. Our data indicates that the assay can detect as little as 100 pM BoNT/A activity within living cells, and the assay is currently being evaluated for the analysis of BoNT in food matrices. Among available in vitro assays, we believe that cell-based assays are widely applicable in high-throughput screenings and have the potential to at least reduce and refine animal assays if not replace it.
Clostridium botulinum is a pathogen of concern for low-acid canned foods. Here we report draft genomes of a neurotoxin-producing C. botulinum strain isolated from water samples used for cooling low-acid canned foods at a canning facility. The genome sequence confirmed that this strain belonged to C. botulinum serotype B1, albeit with major differences, including thousands of unique single nucleotide polymorphisms (SNPs) compared to other genomes of the same serotype.
Facile laboratory tools are needed to augment identification in contamination events to trace the contamination back to the source (traceback) of Salmonella enterica subsp. enterica serovar Enteritidis (S. Enteritidis). Understanding the evolution and diversity within and among outbreak strains is the first step towards this goal. To this end, we collected 106 new S. Enteriditis isolates within S. Enteriditis Pulsed-Field Gel Electrophoresis (PFGE) pattern JEGX01.0004 and close relatives, and determined their genome sequences. Sources for these isolates spanned food, clinical and environmental farm sources collected during the 2010 S. Enteritidis shell egg outbreak in the United States along with closely related serovars, S. Dublin, S. Gallinarum biovar Pullorum and S. Gallinarum. Despite the highly homogeneous structure of this population, S. Enteritidis isolates examined in this study revealed thousands of SNP differences and numerous variable genes (n = 366). Twenty-one of these genes from the lineages leading to outbreak-associated samples had nonsynonymous (causing amino acid changes) changes and five genes are putatively involved in known Salmonella virulence pathways. While chromosome synteny and genome organization appeared to be stable among these isolates, genome size differences were observed due to variation in the presence or absence of several phages and plasmids, including phage RE-2010, phage P125109, plasmid pSEEE3072_19 (similar to pSENV), plasmid pOU1114 and two newly observed mobile plasmid elements pSEEE1729_15 and pSEEE0956_35. These differences produced modifications to the assembled bases for these draft genomes in the size range of approximately 4.6 to 4.8 mbp, with S. Dublin being larger (∼4.9 mbp) and S. Gallinarum smaller (4.55 mbp) when compared to S. Enteritidis. Finally, we identified variable S. Enteritidis genes associated with virulence pathways that may be useful markers for the development of rapid surveillance and typing methods, potentially aiding in traceback efforts during future outbreaks involving S. Enteritidis PFGE pattern JEGX01.0004.