Salmonella Genomics and Population Analyses Reveal High Inter- and Intraserovar Diversity in Freshwater.

Freshwater can support the survival of the enteric pathogen Salmonella, though temporal Salmonella diversity in a large watershed has not been assessed. At 28 locations within the Susquehanna River basin, 10-liter samples were assessed in spring and summer over 2 years. Salmonella prevalence was 49%, and increased river discharge was the main driver of Salmonella presence. The amplicon-based sequencing tool, CRISPR-SeroSeq, was used to determine serovar population diversity and detected 25 different Salmonella serovars, including up to 10 serovars from a single water sample. On average, there were three serovars per sample, and 80% of Salmonella-positive samples contained more than one serovar. Serovars Give, Typhimurium, Thompson, and Infantis were identified throughout the watershed and over multiple collections. Seasonal differences were evident: serovar Give was abundant in the spring, whereas serovar Infantis was more frequently identified in the summer. Eight of the ten serovars most commonly associated with human illness were detected in this study. Crucially, six of these serovars often existed in the background, where they were masked by a more abundant serovar(s) in a sample. Serovars Enteritidis and Typhimurium, especially, were masked in 71 and 78% of samples where they were detected, respectively. Whole-genome sequencing-based phylogeny demonstrated that strains within the same serovar collected throughout the watershed were also very diverse. The Susquehanna River basin is the largest system where Salmonella prevalence and serovar diversity have been temporally and spatially investigated, and this study reveals an extraordinary level of inter- and intraserovar diversity.IMPORTANCESalmonella is a leading cause of bacterial foodborne illness in the United States, and outbreaks linked to fresh produce are increasing. Understanding Salmonella ecology in freshwater is of importance, especially where irrigation practices or recreational use occur. As the third largest river in the United States east of the Mississippi, the Susquehanna River is the largest freshwater contributor to the Chesapeake Bay, and it is the largest river system where Salmonella diversity has been studied. Rainfall and subsequent high river discharge rates were the greatest indicators of Salmonella presence in the Susquehanna and its tributaries. Several Salmonella serovars were identified, including eight commonly associated with foodborne illness. Many clinically important serovars were present at a low frequency within individual samples and so could not be detected by conventional culture methods. The technologies employed here reveal an average of three serovars in a 10-liter sample of water and up to 10 serovars in a single sample.

Salmonella enterica colonization and fitness in pre-harvest cantaloupe production.

Cantaloupes have emerged as significant vehicles of widespread foodborne illness outbreaks caused by bacterial pathogens, including Salmonella. The purpose of this study was to investigate the efficiency of Salmonella colonization and internalization in cantaloupes by relevant routes of contamination. Cantaloupe plants (Cucumis melo 'reticulatus') from two cultivars 'Athena' (Eastern) and 'Primo' (Western) were grown from commercial seed. Plants were maintained in the NCSU BSL-3P phytotron greenhouse. Salmonella enterica (a cocktail of cantaloupe-associated outbreak serovars Javiana, Newport, Panama, Poona and Typhimurium) contamination was introduced via blossoms or soil at ca. 4.4 log10 CFU/blossom or 8.4 log10 CFU/root zone, respectively. Cantaloupes were analyzed for Salmonella by enrichment in accordance with modified FDA-BAM methods. Five randomly chosen colonies from each Salmonella-positive sample were typed using the Agilent 2100 bioanalyzer following multiplex PCR. Data were analyzed for prevalence of contamination and serovar predominance in fruit, stems and soil. Of the total cantaloupe fruit harvested from Salmonella-inoculated blossoms (n = 63), 89% (56/63) were externally contaminated and 73% (46/63) had Salmonella internalized into the fruit. Serovar Panama was the most commonly isolated from the surface of fruit while S. Panama and S. Poona were the most prevalent inside the fruit. When soil was inoculated with Salmonella at one day post-transplant, 13% (8/60) of the plants were shown to translocate the organism to the lower stem (ca. 4 cm) by 7 days post-inoculation (dpi). We observed Salmonella persistence in the soil up to 60 dpi with S. Newport being the predominant serovar at 10 and 20 dpi. These data demonstrate that contaminated soil and blossoms can lead to Salmonella internalization into the plant or fruit at a relatively high frequency.

Plant-Microbe and Abiotic Factors Influencing Salmonella Survival and Growth on Alfalfa Sprouts and Swiss Chard Microgreens.

Microgreens, like sprouts, are relatively fast-growing products and are generally consumed raw. Moreover, as observed for sprouts, microbial contamination from preharvest sources may also be present in the production of microgreens. In this study, two Salmonella enterica serovars (Hartford and Cubana), applied at multiple inoculation levels, were evaluated for survival and growth on alfalfa sprouts and Swiss chard microgreens by using the most-probable-number (MPN) method. Various abiotic factors were also examined for their effects on Salmonella survival and growth on sprouts and microgreens. Community-level physiological profiles (CLPPs) of sprout/microgreen rhizospheres with different levels of S. enterica inoculation at different growth stages were characterized by use of Biolog EcoPlates. In the seed contamination group, the ability of S. enterica to grow on sprouting alfalfa seeds was affected by both seed storage time and inoculation level but not by serovar. However, the growth of S. enterica on Swiss chard microgreens was affected by serovar and inoculation level. Seed storage time had little effect on the average level of Salmonella populations in microgreens. In the irrigation water contamination group, the growth of Salmonella on both alfalfa sprouts and microgreens was largely affected by inoculation level. Surprisingly, the growth medium was found to play an important role in Salmonella survival and growth on microgreens. CLPP analysis showed significant changes in the microbial community metabolic diversity during sprouting for alfalfa sprouts, but few temporal changes were seen with microgreens. The data suggest that the change in rhizosphere bacterial functional diversity was dependent on the host but independent of Salmonella contamination.IMPORTANCE Sprouts and microgreens are considered "functional foods," i.e., foods containing health-promoting or disease-preventing properties in addition to normal nutritional values. However, the microbial risk associated with microgreens has not been well studied. This study evaluated Salmonella survival and growth on microgreens compared to those on sprouts, as well as other abiotic factors that could affect Salmonella survival and growth on microgreens. This work provides baseline data for risk assessment of microbial contamination of sprouts and microgreens. Understanding the risks of Salmonella contamination and its effects on rhizosphere microbial communities enables a better understanding of host-pathogen dynamics in sprouts and microgreens. The data also contribute to innovative preventive control strategies for Salmonella contamination of sprouts and microgreens.

Cluster M mycobacteriophages Bongo, PegLeg, and Rey with unusually large repertoires of tRNA isotypes.

UNLABELLED: Genomic analysis of a large set of phages infecting the common host Mycobacterium smegmatis mc(2)155 shows that they span considerable genetic diversity. There are more than 20 distinct types that lack nucleotide similarity with each other, and there is considerable diversity within most of the groups. Three newly isolated temperate mycobacteriophages, Bongo, PegLeg, and Rey, constitute a new group (cluster M), with the closely related phages Bongo and PegLeg forming subcluster M1 and the more distantly related Rey forming subcluster M2. The cluster M mycobacteriophages have siphoviral morphologies with unusually long tails, are homoimmune, and have larger than average genomes (80.2 to 83.7 kbp). They exhibit a variety of features not previously described in other mycobacteriophages, including noncanonical genome architectures and several unusual sets of conserved repeated sequences suggesting novel regulatory systems for both transcription and translation. In addition to containing transfer-messenger RNA and RtcB-like RNA ligase genes, their genomes encode 21 to 24 tRNA genes encompassing complete or nearly complete sets of isotypes. We predict that these tRNAs are used in late lytic growth, likely compensating for the degradation or inadequacy of host tRNAs. They may represent a complete set of tRNAs necessary for late lytic growth, especially when taken together with the apparent lack of codons in the same late genes that correspond to tRNAs that the genomes of the phages do not obviously encode. IMPORTANCE: The bacteriophage population is vast, dynamic, and old and plays a central role in bacterial pathogenicity. We know surprisingly little about the genetic diversity of the phage population, although metagenomic and phage genome sequencing indicates that it is great. Probing the depth of genetic diversity of phages of a common host, Mycobacterium smegmatis, provides a higher resolution of the phage population and how it has evolved. Three new phages constituting a new cluster M further expand the diversity of the mycobacteriophages and introduce novel features. As such, they provide insights into phage genome architecture, virion structure, and gene regulation at the transcriptional and translational levels.

bettercallsal: better calling of Salmonella serotypes from enrichment cultures using shotgun metagenomic profiling and its application in an outbreak setting.

Most current Salmonella subtyping analyses rely on whole genome sequencing (WGS), which focuses on the high-resolution analysis of single genomes or multiple single genomes from the isolated colonies on microbiological agar plates. In this study, we introduce bioinformatics innovations for a metagenomic outbreak response workflow that accurately identifies multiple Salmonella serovars at the same time. bettercallsal is one of the first analysis tools to identify multiple Salmonella enterica serotypes from metagenomic or quasi-metagenomic datasets with high accuracy, allowing these isolate-independent methods to be incorporated into surveillance and root cause investigations. It was tested on an in silico benchmark dataset comprising 29 unique Salmonella serovars, 46 non-Salmonella bacterial genomes, and 10 viral genomes at varying read depths and on previously well-characterized and sequenced non-selective primary and selective enrichments of papaya and peach samples from separate outbreak investigations that resulted in the identification of multiple Salmonella serovars using traditional isolate culturing and WGS as well as nucleic acid assays. Analyses were also conducted on these datasets using a custom-built k-mer tool, SeqSero2, and Kallisto to compare serotype calling to bettercallsal. The in silico dataset analyzed with bettercallsal achieved the maximum precision, recall, and accuracy of 100, 83, and 94%, respectively. In the papaya outbreak samples, bettercallsal identified the presence of multiple serovars in agreement with the Luminex® xMAP assay results and also identified more serovars per sample, as evidenced by NCBI SNP clustering. In peach outbreak samples, bettercallsal identified two serovars in concordance with k-mer analysis and the Luminex xMAP assay. The genome hit reported by bettercallsal clustered with the chicken isolate genome, as reported by the FDA peach outbreak investigation from sequenced isolates (WGS). Overall, bettercallsal outperformed k-mer, Seqsero2, and Kallisto in identifying multiple serovars from enrichment cultures using shotgun metagenomic sequencing.

Colonization and Internalization of Salmonella enterica and Its Prevalence in Cucumber Plants.

Consumption of cucumbers (Cucumis sativus var. sativus) has been linked to several foodborne outbreaks involving Salmonella enterica. The purpose of this work was to investigate the efficiency of colonization and internalization of S. enterica into cucumber plants by various routes of contamination. Produce-associated outbreak strains of Salmonella (a cocktail of serovars Javiana, Montevideo, Newport, Poona, and Typhimurium) were introduced to three cultivars of cucumber plants (two slicing cultivars and one pickling) via blossoms (ca. 6.4 log10 CFU/blossom, 4.5 log10 CFU/blossom, or 2.5 log10 CFU/blossom) or soil (ca. 8.3 log10 CFU/root zone) and were analyzed for prevalence of Salmonella contamination (internal and external) and serovar predominance in fruit and stems. Of the total slicing fruit harvested from Salmonella-inoculated blossoms (ca. 6.4, 4.5, or 2.5 log10 CFU/blossom), 83.9% (47/56), 81.4% (48/59) or 71.2% (84/118) were found colonized and 67.9% (38/56), 35.6% (21/59) or 22.0% (26/118) had Salmonella internalized into the fruit, respectively. S. Poona was the most prevalent serovar isolated on or in cucumber fruits at all inoculation levels. When soil was inoculated at 1 day post-transplant (dpt), 8% (10/120) of the plants were shown to translocate Salmonella to the lower stem 7 days post-inoculation (dpi). Results identified blossoms as an important route by which Salmonella internalized at a high percentage into cucumbers, and S. Poona, the same strain isolated from the 2015 outbreak of cucumbers imported from Mexico, was shown to be well-adapted to the blossom niche.

Genomics of foodborne pathogens for microbial food safety.

Whole genome sequencing (WGS) has been broadly used to provide detailed characterization of foodborne pathogens. These genomes for diverse species including Salmonella, Escherichia coli, Listeria, Campylobacter and Vibrio have provided great insight into the genetic make-up of these pathogens. Numerous government agencies, industry and academia have developed new applications in food safety using WGS approaches such as outbreak detection and characterization, source tracking, determining the root cause of a contamination event, profiling of virulence and pathogenicity attributes, antimicrobial resistance monitoring, quality assurance for microbiology testing, as well as many others. The future looks bright for additional applications that come with the new technologies and tools in genomics and metagenomics.

Cluster K mycobacteriophages: insights into the evolutionary origins of mycobacteriophage TM4.

Five newly isolated mycobacteriophages--Angelica, CrimD, Adephagia, Anaya, and Pixie--have similar genomic architectures to mycobacteriophage TM4, a previously characterized phage that is widely used in mycobacterial genetics. The nucleotide sequence similarities warrant grouping these into Cluster K, with subdivision into three subclusters: K1, K2, and K3. Although the overall genome architectures of these phages are similar, TM4 appears to have lost at least two segments of its genome, a central region containing the integration apparatus, and a segment at the right end. This suggests that TM4 is a recent derivative of a temperate parent, resolving a long-standing conundrum about its biology, in that it was reportedly recovered from a lysogenic strain of Mycobacterium avium, but it is not capable of forming lysogens in any mycobacterial host. Like TM4, all of the Cluster K phages infect both fast- and slow-growing mycobacteria, and all of them--with the exception of TM4--form stable lysogens in both Mycobacterium smegmatis and Mycobacterium tuberculosis; immunity assays show that all five of these phages share the same immune specificity. TM4 infects these lysogens suggesting that it was either derived from a heteroimmune temperate parent or that it has acquired a virulent phenotype. We have also characterized a widely-used conditionally replicating derivative of TM4 and identified mutations conferring the temperature-sensitive phenotype. All of the Cluster K phages contain a series of well conserved 13 bp repeats associated with the translation initiation sites of a subset of the genes; approximately one half of these contain an additional sequence feature composed of imperfectly conserved 17 bp inverted repeats separated by a variable spacer. The K1 phages integrate into the host tmRNA and the Cluster K phages represent potential new tools for the genetics of M. tuberculosis and related species.