Salmonella Genomics in Public Health and Food Safety PDF

DOMAIN 6 EVOLUTION AND GENOMICS Salmonella Genomics in Public Health and Food Safety ERIC W. BROWN,a REBECC...

DOMAIN 6 EVOLUTION AND GENOMICS Salmonella Genomics in Public Health and Food Safety ERIC W. BROWN,a REBECCA BELL,a GUODONG ZHANG,a RUTH TIMME,a JIE ZHENG,a THOMAS S. HAMMACK,a AND MARC W. ALLARDa a Center for Food Safety and Applied Nutrition, U.S. Food and Drug Administration, College Park, Maryland, USA ABSTRACT The species Salmonella enterica comprises over 2,600 sero- vars, many of which are known to be intracellular pathogens of mammals, birds, and reptiles. It is now apparent that Salmonella is a highly adapted environmental microbe and can readily persist in a number of environ- mental niches, including water, soil, and various plant (including produce) species. Much of what is known about the evolution and diversity of non- typhoidal Salmonella serovars (NTS) in the environment is the result of the rise of the genomics era in enteric microbiology. There are over 340,000 Received: 18 February 2021 Salmonella genomes available in public databases. This extraordinary Accepted: 16 March 2021 breadth of genomic diversity now available for the species, coupled with Published: 14 June 2021 widespread availability and affordability of whole-genome sequencing Editor: David Rasko, University of Maryland (WGS) instrumentation, has transformed the way in which we detect, dif- School of Medicine, Baltimore, Maryland, USA ferentiate, and characterize Salmonella enterica strains in a timely way. Citation: Brown EW, Bell R, Zhang G, Timme Not only have WGS data afforded a detailed and global examination of R, Zheng J, Hammack TS, Allard MW. 2021. the molecular epidemiological movement of Salmonella from diverse envi- Salmonella genomics in public health and ronmental reservoirs into human and animal hosts, but they have also food safety. EcoSal Plus 9:eESP-0008-2020. allowed considerable consolidation of the diagnostic effort required to https://doi.org/10.1128/ecosalplus.ESP-0008 test for various phenotypes important to the characterization of -2020. Salmonella. For example, drug resistance, serovar, virulence determinants, Address correspondence to Eric W. Brown, [email protected]. and other genome-based attributes can all be discerned using a genome This is a work of the U.S. Government and is sequence. Finally, genomic analysis, in conjunction with functional and not subject to copyright protection in the phenotypic approaches, is beginning to provide new insights into the pre- United States. Foreign copyrights may apply. cise adaptive changes that permit persistence of NTS in so many diverse and challenging environmental niches. KEYWORDS whole-genome sequencing, foodborne pathogen isolates, data sharing, ontology, Salmonella detection, genomic impact, epidemiology, microbiology, adaptive change, nontyphoidal salmonellae The genus Salmonella represents a group of rod-shaped, Gram-negative, fac- ultatively anaerobic bacteria in the family Enterobacteriaceae. The genus is composed of two species, Salmonella bongori (originally called Salmonella subspecies V) and Salmonella enterica (1). The latter species contains a great number (.2,600) of serologically distinct variants, or serovars, known to persist predominantly in mammals, birds, and reptiles. Taxonomically, the EcoSalPlus.asm.org 1 Brown et al. species Salmonella enterica is partitioned into seven sub- adaptation of Salmonella into food and feed environ- species, including I (Salmonella enterica subsp. enterica), ments. WGS source tracking is allowing us to see novel II (S. enterica subsp. salamae), IIIa (S. enterica subsp. ari- evidence of exposure of fresh-cut produce to foodborne zonae), IIIb (S. enterica subsp. diarizonae), IV (S. enterica pathogens in animal reservoirs and through contaminated subsp. indica), VI (S. enterica subsp. houtenae), and VII water and soil amendments. (2, 3). However, nearly all (.99%) of those serovars associated with clinical and veterinary illness are derived The importance of genetic and genomic data in under- from subspecies I (3). standing the ecological and evolutionary adaptations that drive persistence of foodborne pathogens such as Salmonella is a highly fit environmental microbe, how- Salmonella cannot be overstated. Application of whole- ever, and enjoys a distribution that is largely ubiquitous genome sequencing (WGS) for the characterization of across geographic and biologic reservoirs. The pathogen Salmonella has provided extraordinary insight into epi- is well known to persist frequently outside animals in demiology, biology, evolution, and population structure other natural environments, including fresh and marine of Salmonella over the past decade (9), including per- surface waters, soil, and dust, and as epiphytes on and mitting the detailed organization of Salmonella enterica inside plant materials (4). Since its divergence from into a phylogenetic hierarchy that largely recapitulates Escherichia coli more than 100 million years ago, the species’ natural population structure (10). The WGS Salmonella has undergone widespread evolutionary of Salmonella strains now regularly provides a highly diversification and niche-specific adaptive change reliable and predictive means to ascribe various pheno- through the acquisition of numerous novel genomic typic and diagnostic traits to a specific isolate by means changes, many of which have been acquired as a of one analytical workflow—that is, the sequence itself result of horizontal gene transfer (HGT) and other (9). Historically, during surveillance and diagnostics, reticulate evolutionary forces (3). important phenotypic tests such as serotyping, antimi- crobial resistance (AMR) testing, and phage typing Salmonella enterica is responsible for 1.4 million (PT) were cumbersome and expensive, but genetic and cases of foodborne salmonellosis in the United States genomic alternatives are already developed that can annually, making it the number one causative agent provide comparable and quite reliable results simply of bacterial foodborne illnesses. Infection can occur by analyzing the genomic sequence of a particular after eating undercooked meat, poultry, and eggs, as Salmonella isolate. well as fresh-cut produce that is readily consumed raw and has been contaminated with Salmonella (5). The same genomic approaches to phenotypic discov- In recent years, more Salmonella-related outbreaks ery are also beginning to yield clues regarding the have occurred in the United States associated with emergence of unique and strongly selected adapta- the consumption of produce than animal-based food tions in Salmonella, some of which have transformed commodities. Recent outbreaks of this nature include Salmonella with novel virulence traits and capabilities a massive Salmonella enterica serovar Saintpaul out- in its host as well as permitting it to endure in envi- break associated with tomatoes, jalapeños, and ser- ronmentally harsh and unexpected environments and rano peppers that sickened over 1,400 individuals in to persist in the face of otherwise lethal assaults from 2007 (6) as well as four separate events involving antimicrobials, oxidative agents, and other sanitizers Maradol papaya in 2017 that included at least 8 dif- in industrial and health care settings. This has ferent serovars and caused more than 250 known ill- become particularly true in the food production and nesses, including two deaths (7). Additionally, a 2014 processing industry, where recent genetic adaptations cucumber-related outbreak associated with Salmonella observed in Salmonella may subvert certain controls enterica serovar Newport and Salmonella enterica serovar and preventions and contribute to foodborne illness Javiana caused more than 275 reported illnesses and one of public health concern. One example of this death (8). These events underscore the notion that numer- includes a recent outbreak strain of Salmonella enter- ous serovars of Salmonella may have migrated successfully ica serovar Bareilly isolated from tuna, which was into previously naive niches (i.e., produce-growing niches) found to harbor a genomic island containing a previ- and point to a role for the ongoing genetic and epigenetic ously undescribed arsenic resistance operon (11, 12). 2 EcoSalPlus.asm.org  Salmonella Genomics in Public Health and Food Safety S. Bareilly isolates that carry the novel genetic island investigation into Salmonella outbreaks and contamina- are significantly more resistance to arsenic than those tion events. The WKL approach uses the agglutination that do not. Arsenic is a toxic metalloid ubiquitous in reaction with antisera against O and H antigenic var- the natural environment and often found at higher iants (15). Currently, 46 O antigens and 114 H antigens levels in fish and seafood, which absorb arsenic from exist among the known salmonellae, and from the pos- surrounding waters and other lower trophic species sible combinations, about 2,600 have been isolated and that are part of their diet. With the novel arsenic resist- named “serovars” of the species (16). Although it was a ance element integrated into the chromosome, Salmonella staple of Salmonella serological characterization for Bareilly is now likely to better survive and persist in tuna. decades, the method rapidly is being replaced by the This highlights the potential impact of evolutionary widespread availability of genomic approaches to sero- change in Salmonella, whereby a selective advantage was typing of the species. Moreover, the schema itself (i) conferred for survival, persistence, and even growth relies heavily on antisera that are now available from a within human food matrices. Moreover, by pinpointing dearth of sources, (ii) depends on the availability of the underlying differences in phenotype among closely highly trained experts to interpret often complex and related salmonellae, we are better able to predict sometimes ambiguous results, and (iii) requires suffi- Salmonella’s environmental responses and subsequently cient time to complete the reactions while remaining in may be able to provide specific and targeted mitigation step with an investigation or traceback event. In order strategies, recently termed “precision food safety” to overcome these shortfalls, various molecular testing- approaches, for controlling nontyphoidal Salmonella sero- based strategies were developed in the early 2000s that vars (NTS) from further contaminating the food and feed used gene sequences as surrogate antigenic markers, supply (13). including multiple PCR-based approaches (17–19) and a liquid suspension DNA hybridization approach based Continued understanding of Salmonella fitness, diversi- on X-map (e.g., Luminex) technology (20, 21). fication, virulence, and survivability will be essential to our ability to manage, treat, and prevent its contamina- More recently, genomic approaches to Salmonella sero- tion of humans and human-associated upstream niches. typing have risen in popularity and utility. An abun- While much remains to be discerned regarding the dance of genomic data from the species is now widely ever-changing face of Salmonella in its natural habitat, available for comparison of WGS-derived phenotypes, it is now clear that Salmonella microbiology has already including serovar status (12). Several of these methods been advanced tremendously from the information pro- now hold great potential for deployment and use by vided by genomic tools and in particular the integration public health and food safety authorities, including the of WGS into traditional microbiological areas of the U.S. FDA, the CDC, Public Health England, and Public study of Salmonella (9). Here, it is our intent to present Health Canada, to name a few. the impact of genomics in several key areas of Salmonella microbiology, including its phylogenetic An early example of predicting serovar was a WGS- partitioning, adaptive changes, environmental persist- based solution called SeqSero (22). This genomic dash- ence, host specificity, virulence, and continued burden board tool, launched in 2015, relies solely on the upload on food and feed safety. of a draft Salmonella genome to the SeqSero web tool (http://www.denglab.info/SeqSero) and subsequent receipt of the genomic serotype of the Salmonella strain based on TRANSFORMATION OF SALMONELLA the rapid genomic comparison of O and H antigen-encod- SEROLOGY AND SEROTYPING THROUGH ing genes. Very recently, an enhanced functional update WGS and new version of the software, SeqSero2 (https://github Serotyping has long been a key classification for.com/denglab/SeqSero2/branches) (16), was released Salmonella. The White-Kauffmann-Le Minor (WKL) that is 50 faster than its predecessor with a serovar call scheme is the international standard for the designation accuracy of 98% when evaluated against several large of Salmonella serotypes and is based on serological Salmonella WGS-based public databases, including National characterization of the O and H antigens (14). Antimicrobial Resistance Monitoring System (NARMS) and Serotyping remains a critical part of public health the GenomeTrakr National databases. An additional EcoSalPlus.asm.org 3 Brown et al. genome-based typing tool was also recently developed Numerous genetic and genomic studies on population that targets the serovar-specific spacer regions of the two structure and chromosome organization in Salmonella CRISPR loci (i.e., CRISPR 1 and CRISPR 2) in Salmonella. have repeatedly demonstrated that HGT has driven the The method, called CRISPR-SeroSeq, provides a multi- emergence of highly adapted strains of Salmonella in the plexed partial genome-sequencing scheme that can environment, on the farm, and in the food supply (25). detect and characterize multiple Salmonella serovars While numerous mechanisms and pressures can drive from a single analysis. This direct serotyping approach HGT among the salmonellae, the hypermutable phenotype can pick up underrepresented serovars in a single envi- has underscored numerous examples of reticulate evolu- ronmental sample as low as 0.01% (23), making it partic- tion on the Salmonella chromosome. Methyl-directed mis- ularly attractive for direct serotyping of poultry and match repair (MMR) defects, leading to the “mutator,” or poultry processing related swab surveillance samples, hypermutable, phenotype, are found in more than 1% of where numerous serovars can populate an individual test the isolates within naturally occurring populations of S. (24). The software improvements are supported by large enterica and at even greater frequencies in the food supply, curated databases of more known serovar sequences to where oxidative and other antimicrobial stressors often compare against new uploaded unknowns. This strategy abound (26). Up to 73% of the MMR defects found in feral of building curated known reference sequence databases settings are the result of lesions within mutS, resulting in in specific bioprojects will allow investigators to build increased nucleotide substitution rates, enhanced DNA more genomic tools that predict phenotype from geno- transposition, and, perhaps most importantly, a relaxation type for any genes that are fully characterized and linked of the internal barriers that normally restrict homeologous to specific phenotypes. recombination following the horizontal acquisition of for- eign DNA (27–30). Phylogenetic analyses were conducted of the Salmonella reference (SAR) collections (i.e., SARA, RECOMBINATION, RETICULATE SARB, and SARC), which were largely considered to rep- EVOLUTION, AND THE IMPACT OF HGT IN resent the extent of genetic variability within the species SALMONELLA but are now known to represent a subset of that diversity Salmonella evolves both vertically and horizontally. By (2, 31, 32). Work first suggested by single-gene studies vertical evolution, we mean ancestor-to-descendant evolu- revealed striking levels of phylogenetic discordance tion based on passing genetic variants on to the next gen- between trees derived from mutS alleles and whole-chro- eration through inherited genetic changes. In contrast, mosome trees of the same strains based on multilocus horizontal evolution refers to genetic variation that was enzyme electrophoresis (MLEE) analysis (28, 29). These not inherited but rather exchanged between organisms findings support the notion that HGT helped forge cur- (HGT). Plasmids and phages can be transferred through rent relationships among Salmonella and other enteric conjugation to other compatible bacteria. Thus, bacterial pathogens in this region and throughout numerous other isolates that independently acquired horizontal elements locales in the Salmonella chromosome. Indeed, as evi- (phages and plasmids) may appear closely related when denced in other studies employing genomic scanning they are distantly related and share only the HGT ele- approaches, such as WGS, microarray analysis, and ments. HGT may confound vertical evolution; that is why multilocus sequence typing (MLST), the substantial these potential elements are initially filtered out when impact that HGT has had on structuring the chromo- building phylogenetic trees to document closely related some of Salmonella enterica is indisputable (33, 34). isolates that share an ancestor. Variation from recombina- Estimates based on such studies indicate that more tion also may confound vertical evolution and thus also than one-quarter of the Salmonella genome may have may be filtered out to more accurately measure the verti- been brought about by HGT and reticulate evolution- cal evolutionary signal, as the focus for WGS in food ary forces (33), although this number is likely conserv- safety is to identify the most closely related isolates and to ative, based on current views. cluster them for follow-up investigation. Phylogenetic comparisons between different regions of the Salmonella It is now evident that HGT has played a key role in genome cemented the key role of HGT in the genetic and structuring many other regions of the Salmonella chro- evolutionary diversification of S. enterica subspecies, sero- mosome as well. Notably, Salmonella pathogenicity vars, and individual pathogenic clones. islands (SPI) were likely acquired through HGT (35–37). 4 EcoSalPlus.asm.org  Salmonella Genomics in Public Health and Food Safety For example, SPI-1, comprising the genes encoding a between the most closely related salmonellae (29). To type III secretion system, was probably acquired early in this end, phylogenetic studies also have revealed the of- Salmonella evolution (38), yet several inv-spa alleles seem ten-underappreciated role of HGT and recombination to have converged horizontally more recently between S. in the homogenization of allele structure within closely enterica groups IV and VII (28, 39). Additionally, genes related populations of S. enterica (40), as well as a ge- comprising the inv-spa gene cluster appear to have netically panmictic structure for restriction-modifica- undergone extensive allelic shuffling among the group I tion (R-M) genes among group I salmonellae. The latter salmonellae (40). Also, type 1 pilin genes, encoding fimb- finding, noting open exchange of R-M (i.e., hsd) alleles, rial adhesins, retain unusually low GC content and constitutes phylogenetic evidence of the compatibility of obscured phylogenies relative to other fim genes (41). S. enterica subspecies I R-M complexes, likely accounting Other studies focusing on housekeeping genes in for the documented successful HGT of entire gene Salmonella have reported evolutionary histories for sequences among closely (e.g., intrasubspecies) related these genes that are strikingly decoupled from S. strains, as DNA exchange between strains that share or enterica strain history (28, 42–44). recently shared common R-M alleles would not be sub- ject to substantial restriction (50). In comparing across and within subspecies of Salmonella, a recombination “gradient” has been noted wherein lateral The phylogenetic data demonstrate that HGT has been DNA transfer appears to be inversely correlated with a frequent and regular phenomenon among the salmo- genetic variability among strains. Subsequently, a genetic nellae, and this may explain, in part, why Salmonella boundary may exist that tolerates free recombination of possesses such unique evolutionary histories for numer- DNA sequences within a framework delimited by sequence ous genes, operons, and islands within its chromosome. variation and niche diversity of individual Salmonella With the complete genome sequences of literally hun- strains. This has been documented previously through the dreds of thousands of Salmonella isolates now available, observation of intragenic (or patch-like) recombination such refined genomic and phylogenetic analyses should events among more diverged Salmonella subspecies and aid in arriving at the final verdict on the impact of assortative (whole-allele) recombination events, responsible recombination on Salmonella chromosome structure. for extensive whole-allele reassortment, among more genet- Certainly, a greater recognition of precisely how HGT ically homogeneous populations of group I Salmonella and homeologous and homologous recombination have strains (28, 29). It is notable that the latter strains all share a forged the genomes of Salmonella pathogens should niche primarily found in warm-blooded mammals (2). enhance the accuracy of our risk assessment of these pathogens as well as providing solutions for better Largely due to the recent availability of genomic data, detection and characterization of this devastating food- our understanding in reconstructing the HGT of im- borne disease-causing agent. portant features, including those involved in virulence, drug resistance, and other adaptations that foster an enhanced fitness for Salmonella persistence in the farm- THE IMPACT OF GENOMICS ON THE to-fork continuum is expanding at a pace that we could DETECTION AND CHARACTERIZATION OF not have foreseen at the turn of the millennium (12). AMR IN SALMONELLA The extent and effects of recombination have now been The AMR S. enterica strains are a significant source of noted for both typhoid-causing salmonellae and NTS in enteric foodborne illness and a public health concern important clinical and environmental niches and across (51). In particular, the presence of AMR genes associated both core genome regions and the Salmonella mobilome with NTS in the food and feed supply presents an impor- (45–47), including AMR determinants, a suite of chemi- tant challenge to controlling human and veterinary ill- cal resistance operons (48), and numerous Salmonella ness associated with the consumption of contaminated genomic island (SGI) regions (49). food and feed commodities (52, 53). The AMR genes are found across large numbers of Salmonella genomes, with It is important to recall, however, that reticulate evolu- comparable averages for Salmonella associated with pro- tionary pressures do not subside once selectively advan- duce and animal food products of 72% and 74%, respec- tageous traits are gained. Rather, HGT likely continues tively (54). This high prevalence of AMR genes has been EcoSalPlus.asm.org 5 Brown et al. attributed specifically to an elevated presence of amino- is generally limited to a smaller subset of antibiotics, glycoside resistance genes along with tetracycline and whereas a genomic screen interrogates all known AMR sulfonamide genes, in most food isolates (54). genes supported in the database. A search of the NCBI Pathogen Detection website identifies a Salmonella ge- An automated AMR typing approach that relies on nome with up to 29 AMR genes present and over 200 genomic data has been developed to meet the challenge isolates with up to 20 AMR genes present. The of AMR characterization in Salmonella associated with AMRFinderPlus database has been expanded to predict the food supply. Consistent with other WGS tools now genes related to stress and virulence. These publicly available for predicting phenotype from genotype, AMR available reference databases allow any investigator to genotypes can now be readily targeted from WGS and create rapid PCR or sequencing panel screens for specific the resultant phenotype predicted (55). The importance genes of particular interest to stakeholders. This is a of these Salmonella AMR tests cannot be overstated, as strategy to reduce the costs of genotype-to-phenotype tens of thousands are conducted each year by the federal predictions by targeting gene panels for food safety, government and their public health partners, primarily industry surveillance, and clinical diagnostics. through the NARMS program (https://www.fda.gov/ animalveterinary/safetyhealth/antimicrobialresistance/ nationalantimicrobialresistancemonitoringsystem/), a Salmonella found in the environment, food, and consortium of state and federal agencies that monitor animals: adaptive fitness and persistence. Two AMR in meats and clinically obtained isolates of types of clinical manifestations were associated with Salmonella and other enteric bacterial foodborne Salmonella serovars, including invasive, life-threatening pathogens. To this end, The National Center for systemic disease, referred to as typhoid fever, and self- Biotechnology Information (NCBI), in collaboration with limited gastroenteritis caused by NTS (56–58) found in experts on AMR in Salmonella, provide online WGS tools foods, animals, and the environment. However, 5% of for predicting AMR genotypes (https://www.ncbi.nlm.nih individuals infected with NTS develop bacteremia (59),.gov/pathogens/). The NCBI has produced AMRFinder and disease manifestations are substantially different Plus, a tool that identifies AMR genes using a high-quality between different serovars (60–62). Multiple NTS were curated AMR gene reference database (55). The Bacterial found in asymptomatic food-producing livestock, includ- AMR Reference Gene Database consists of up-to-date gene ing poultry, sheep, cattle, and swine (63–65), indicating nomenclature, a set of hidden Markov models (HMMs), that Salmonella persistence and carriage in livestock are and a curated protein family hierarchy. Predictive assess- very common, possibly since E. coli and Salmonella ments of AMRFinderPlus revealed genotype-to-phenotype diverged from a common ancestor. Multiple sets of concordance for Salmonella AMR profiles of more than Salmonella genes are involved in prolonged infection and 98%, pointing to a margin of error of less than 2% and pro- persistence (66). Distinct sets of fimbriae contribute to the viding evidence that AMRFinderPlus is a highly accurate intestinal persistence and colonization in different animal WGS-based AMR gene detection system (55) for each draft species (67, 68). Besides fimbrial adhesins, other adhesins, genome uploaded and released through the WGS pathogen including the autotransporter adhesins MisL, SadA, and portal at NCBI. The NCBI Pathogen Detection web site ShdA, the type I secretion system-secreted adhesins SiiE also provides investigators with detailed guidance on how and BapA, and curli biogenesis (csg) adhesins, were found to upload corresponding phenotypic antibiogram metadata to play a role in colonization and persistence in mouse with their draft genomic data so that improved calling of gastrointestinal tract (69–73). Recent works suggested that AMR genotypes can ensue. biofilm formation is involved in Salmonella gallbladder persistence. Salmonella gallbladder colonization triggers However, other questions surrounding AMR salmonellae upregulation of the O-antigen capsule-encoding operon remain, including those concerning the geographic dis- (yihU-yshA and yihV-yihW) in an agfD-independent tribution of AMR genotypes (see the FDA CVM manner, which is specifically required for biofilm forma- Resistome Tracker), the genomic diversity present in tion on cholesterol gallstones (74). Iron is an essential nu- known AMR genes, and how much differential expres- trient for human and animals. A major host defense sion and phenotypic variation is present in known genes against infection is nutritional immunity, e.g., via seques- and allelic AMR variants. Traditional antibiogram testing tration of metals, including iron (75), to prevent pathogen 6 EcoSalPlus.asm.org  Salmonella Genomics in Public Health and Food Safety growth (76). The siderophore ABC transporter FepBDGC enterica serovar Choleraesuis and Salmonella enterica is responsible for primary ferric ion import in Salmonella. serovar Dublin, cause severe systemic disease in their It was shown that the fep system together with the ferric- natural host and humans which is also characterized iron-binding siderophores enterobactin and salmochelin by fever and septicemia with little diarrheal symp- is required for persistent Salmonella infection in mice toms (82). This is in contrast to most Salmonella (77). Comparative genomic analysis of serial isolates asso- serotypes, which exhibit a broad or unrestricted host ciated with long-term epidemics revealed mutation rates range and cause severe gastroenteritis. In recent from 1.9  1027 substitution site21 year21 to 1.49  1026 years, though, there has been an emergence of HA in substitution site21 year21 in the core genome of certain clones of some unrestricted-host-range sero- Salmonella enterica serovar Typhimurium (78–80). Multiple types which cause invasive disease mainly in immu- nonsynonymous single nucleotide polymorphisms (SNPs) nocompromised patients (Table 1) (83, 84). were found in global virulence regulators, including DksA, RpoS, HilD, MelR, and BarA, and metabolic pathways, The precise mechanisms of how HA and HR evolved providing an adaptative advantage during persistence in are not well understood but seem to encompass three the host (79, 81). Meanwhile, the mean genome-wide rate major steps: the gain of genetic information, the loss of of nonsynonymous to synonymous substitutions (dN/dS) genetic information (genome reduction), and the was less than 1 during the short-term evolution of increase of pseudogenes within the genome. During the Salmonella, indicating that the underlying substitution evolution of HR, S. Typhi gained several pathogenicity rate is subject to purifying selection (78). In contrast to islands (SPI-7, -15, -17, and -18), which includes the Vi the relatively stable core genome, considerable variation in capsular antigen, which allows it to avoid being killed composition of mobile genetic elements, including pro- by host complement and prevents phagocytosis (85). phages and plasmids, was identified within the same clone Conversely, S. Typhi and other HA/HR serovars tend to in the course of an epidemic (78–81). All these changes be auxotrophic for specific amino acids and vitamins contributed to clinically relevant differences in phenotype (82) and have lost large numbers of genes in anaerobic and virulence, further emphasizing the critical importance metabolic pathways necessary for growth in the inflamed of integrated genotypic data sets in understanding of bio- gut (86). Also, many have a reduced number of virulence logical variability in Salmonella epidemiology. factors commonly found in broad-host-range serovars (87). Along with the loss of genes, all HA and HR sero- vars have large numbers of pseudogenes compared to Host adaptation. Primarily, when infecting its host, unrestricted-host-range serovars (85–88). Many of the Salmonella exists in the intestinal tract as a gastrointesti- genes seen to be degraded in HR and HA serovars tend nal pathogen with limited duration and disease progres- to be involved in motility or chemotaxis, to encode type sion. However, some serovars have adapted to cause an III secretion effectors, or to encode structures involved invasive disseminated disease. During this process, these in attachment to host cells, such as fimbriae and other serovars also have lost the ability to infect a broad range adhesins (85). In addition, the allelic variation found of hosts, becoming much more host adaptive (HA) or within the HA and HR group is reduced compared to host restrictive (HR). Host-adaptive serovars tend to host generalists, suggesting their more recent emergence have one or two main animals that they naturally infect in specialized host species (87). Allelic variation also but are capable of infecting other hosts given the oppor- likely has played a role in the host tropism in salmonel- tunity. Host-restricted serovars have one main host and lae, which may be a first step toward host restriction. For rarely or never naturally infect a different host. Well- example, Yue and colleagues studied allelic diversity of known examples of HR serovars include Salmonella several fimbrial adhesins and found patterns of alleles enterica serovar Typhi, Salmonella enterica serovar associated with different host types (89–91). Paratyphi A, Salmonella enterica serovar Gallinarum/ Pullorum, and Salmonella enterica serovar Abortusovis Some clones of Salmonella are adapting to a more re- (Table 1) (82). The disease caused by these serovars in stricted host range. Two prominent examples of this their natural host is typically characterized by fever include invasive S. Typhimurium ST313 and invasive and septicemia, with very little or no gastrointestinal Salmonella enterica serovar Enteritidis, which have disease. Similarly, HA serovars, such as Salmonella emerged in sub-Saharan Africa (83, 84). Genetic analysis EcoSalPlus.asm.org 7 Brown et al. TABLE 1 Characteristics of host-restricted, host-adapted, and unrestricted invasive Salmonella serovarsa Missing genes/ Serovar Host Auxotrophic requirementb Disease pseudogenes Host-restricted serovarsc Typhi Humans, Tryptophan, cobalamin Typhoid fever sseI, gtgE, sopA, sseK2, ratB, chimpanzees (vitamin B12) sadA, stfH, sopD2, gtgA, ompD, steB, sopE2, shdA, sinH, bapA, avrA, misL, cigR, sseK1 Paratyphi A Humans Cystine, arginine, Enteric fever cobalamin (vitamin B12) Gallinarum/Pullorum Poultry Cystine, leucine, aspartic Fowl typhoid, Pullorum sseI, gtgE, sopA, sseK2, ratB, acid, thiamine, cobalamin disease sadA, sirP, sifB, fliC, sspH2 (vitamin B12) Abortusovis Ovines Cystine, nicotinic acid Abortions, newborn mortality (vitamin B12), thiamine Typhisuis Swine Cystine Chronic paratyphoid Abortusequi Equines Abortions, newborn mortality Host-adapted serovarsd Choleraesuis Swine, Swine paratyphoid sseK2, sspH2, shdA, avrA, humans sadA, sopE Dublin Bovines, Nicotinic acid (vitamin B3) enteric and invasive disease, srfN, fliC, sseK2, shdA, humans, abortions bapA, sadA ovines Invasive NTS strain or serovar Typhimurium ST313 Humans Cobalamin (vitamin B12) Invasive disease sseI, shdA, siiE, sspH2, sadA Enteritidis Humans Cobalamin (vitamin B12) Invasive disease sseI, sspH2, shdA, sadA, siiE, fliC, sseK2, sinH a Data are from references 82 and 87. b Auxotrophy is a common characteristic in host-restricted and host-adapted serovars. c Host-restricted disease tends to be systemic with little or no gastroenteritis. d Host-adapted disease does not produce severe enteritis and is followed by systemic dissemination. of S. Typhimurium ST313 has shown signatures of adap- localized to western areas and one to central/eastern areas tation. For example, a number of pseudogenes have been (83). These clades show signatures of adaptation similar to identified in ST313, compared to other S. Typhimurium those of other host-adapted or restricted serovars, namely, strains, and are similar to the pseudogenes found in S. multiple pseudogenes in metabolic pathways along with an Typhi and S. Paratyphi A, including ratB, ttdA, and sseI accumulation of nonsynonymous SNPs in membrane pro- (79, 80, 84). Also similar to S. Typhi and S. Paratyphi A, S. tein genes (83). It should also be noted these invasive sero- Typhimurium ST313 shows a loss in metabolic capacity vars are multidrug resistant (MDR) and have most likely (80). Interestingly, ST313 has a limited ability to form bio- gained the genes for drug resistance since the divergence films, which may lead to reduced fitness to survive outside with their most recent common ancestor (83, 84). the human host (92); this in turn may explain the lack of an identified zoonotic reservoir and evidence for human- to-human spread of this pathogen (93). In parallel, analysis Examples from poultry-adapted serovars. (i) Salmonella of S. Enteritidis isolates linked to invasive disease have enterica serovar Enteritidis. Salmonella Enteritidis is a identified two clades circulating in sub-Saharan Africa, one host-promiscuous serovar that is predominantly 8 EcoSalPlus.asm.org  Salmonella Genomics in Public Health and Food Safety associated with gastroenteritis. Separating from S. serovars (95). Accumulation of different FGCs may Dublin, rather being a single clade itself, S. Enteritidis is improve the efficiency of specific host colonization and more structurally complex. The WGS phylogeny of S. broaden the host range. Comparative genomics of out- Enteritidis suggests the presence of at least four clades break-related bovine strains and isolates from other with three epidemic clades and one clade from which S. resources have predicted that the gain of Saf fimbrial Gallinarum/S. Pullorum complex directly evolved (83, genes may have contributed to the increased bovine 88). Among the three epidemic clades, one is the classic colonization (96). Moreover, S. Heidelberg isolates pre- or global epidemic clade (83, 94) including the most sented divergent MDR genes with strong phylogeo- commonly isolated MLSTs and PTs associated with graphic signature (97) and displayed a broad diversity of enterocolitis in human. The other two S. Enteritidis phage-related genes, with some unique to different poul- clades emerged from Africa and are strongly associated try farms (98). Phage and plasmid HGT may facilitate with multidrug resistance and invasive disease (83). the dissemination of MDR (95, 99, 100) and contribute Given the genetic and phenotypic heterogeneity within to the fitness of S. Heidelberg in different poultry farm S. Enteritidis clades, multiple signatures of differential environments (98). host adaptation are observed in the context of genome evolution. Analysis of the accessory genome, consisting of 14,015 predicted genes, showed the acquisition of a (iii) Salmonella enterica serovar Kentucky. S. Kentucky novel prophage region closely related to Enterobacter is among the S. enterica serovars most frequently iso- phage P88 and an enlarged virulence plasmid with lated from poultry in the United States (101, 102) and acquired MDR genes in both African clades. Additionally, has been increasingly isolated from dairy cattle as well strains from both African lineages harbored pseudogenes (103). However, it is less commonly identified as a which are concentrated in common metabolic pathways, source of human salmonellosis than other serovars as observed in other host-restricted invasive Salmonella commonly detected in poultry, such as S. Enteritidis serovars. Some of the overlaps are astonishing, including and S. Heidelberg. Although S. Kentucky contains five reduced metabolic activity in cobalamin and propanediol pathogenicity islands (SPI-1 to -5), like other serovars utilization and also ornithine decarboxylase activity, indic- in S. enterica, the lack of full-length SPI-2-associated ative of the role of gene loss/pseudogene formation in the genes and fimbrial genes (104, 105) might compromise adaptation from a gut to a systemic lifestyle. its virulence in humans. The rise of S. Kentucky as the dominant serovar in poultry may be due to the acid response phenotype (106) and a metabolic advantage (ii) Salmonella enterica serovar Heidelberg. Salmonella conferred by the acquisition of the ColV plasmid for Heidelberg is primarily a poultry-adapted serovar of scavenging scarce energy sources available in the Salmonella that can colonize and infect multiple hosts. chicken cecum (107). Phylogenetic analysis indicated Infections with S. Heidelberg are more likely to be inva- that S. Kentucky is polyphyletic (57, 108), with two sive and associated with greater risk for severe disease highly divergent ST complexes. ST152 and ST198 are than other serovars (95). The pan-genome (pan, from the most frequently isolated S. Kentucky sequence types the Greek word p a, meaning “whole”) includes a core in each ST complex globally. ST198 is reported to be genome containing genes ubiquitous in all strains and MDR and causes gastroenteritis in humans (109), while an accessory genome composed of genes absent from ST152 is rarely associated with human disease (108). one or more strains and genes that are unique to each Comparative genomics between ST152 and ST198 iso- strain. The pan-genome of S. enterica subspecies I is lates found significant differences in gene content and predicted to have 42 or 43 different fimbrial gene clus- core genome nucleotide sequence divergence. The roles ters (FGCs), which have been implicated in host coloni- of several genomic elements in ST198, such as a sialic zation and adaptation. With acquisition and deletion of acid transport region, inositol catabolism, and a homo- FGCs, the evolutionary pathway has led to four clades log of the Typhi colonization factor, need to be further of S. enterica subspecies I. S. Heidelberg resides in clade evaluated for host-associated colonization (108). It also 1b with two other serovars, Salmonella enterica serovar is noted that MDR is mostly conferred by plasmids in Virchow and Salmonella enterica serovar Hadar, carry- poultry-associated S. Kentucky ST152 isolates (102, ing the highest numbers of FGCs among all other 108), while it is associated with the acquisition of EcoSalPlus.asm.org 9 Brown et al. Salmonella genomic island 1 (SGI1), plasmids, and muta- Heidelberg in poultry populations. The evolution of tions in the core genome of ST198 isolates (108–110). Salmonella genomes related to poultry together with intervention strategies in poultry population marked a path toward the shifts in Salmonella serovars in egg and Patterns of Salmonella serovar evolution in egg and poultry production. poultry production. Egg- and poultry-associated prod- ucts have been frequently implicated in foodborne gas- troenteritis caused by Salmonella serovars (111). Salmonella virulence and genomic evolution. Compared Predominant Salmonella serovars in commercial poul- to Escherichia coli, which has a bigger pan-genome try have undergone significant shifts over the last sev- (127), S. enterica has a smaller pan-genome, which indi- eral decades. Several bacterial factors could contribute cates that the rate of discovery of new genomic regions to such shifts in Salmonella populations in poultry, would decrease for each new genome of the species including competitive exclusion and genetic factors that sequenced (128–130). The S. enterica pan-genome and facilitate Salmonella colonization in poultry. The preva- core genome have been examined based on different lence of Salmonella serovars among poultry can be sets of available genomes (56, 57, 127–132). A recent dated back to the early 1900s. S. Gallinarum biovars study of 4,893 genomes of S. enterica identified a pan- Pullorum and Gallinarum caused pullorum disease and genome of 25.3 Mbp, a strict core of 1.5 Mbp present in fowl typhoid in poultry, respectively, posing a serious all genomes, and a conserved core of 3.2 Mbp found in economic threat to the poultry industry at that time at least 96% of these genomes. Given an average gene (112). In the 1980s, S. Enteritidis O9,12:g,m emerged as size of 1,000 bp, the core genome has ;1,500 genes and a major public health problem in Europe and the includes ;3,200 genes in the conserved core genome, Americas (113). S. Enteritidis did not spread to domes- with a much larger pan-genome of ;25,300 genes tic fowl until much later after the initial introduction (131). Worley et al. (132) described a core genome for into poultry flocks through its rodent animal reservoir Salmonella that included 2,278 genes present only (114–116). S. Enteritidis shared the immunodominant once in each genome and of the same length, without O antigen (O9) on the cell surface with Gallinarum, indels, comprising 2,036,954 bp, which is less than half which may have contributed to the exclusion of S. of the known reference genome of S. enterica serovar Enteritidis earlier (117, 118). One of the reasons for the Typhimurium LT2, comprising 4,857,450 bp. Larger increased spread of S. Enteritidis could be the eradica- Salmonella genomes have been reported. tion of S. Gallinarum, which may have opened an eco- logical niche for S. Enteritidis to fill (119). S. enterica phylogeny based on WGS indicated that important acquisitions from a virulence perspective The prevalence of S. Enteritidis has declined in chicken included acquisition of SPI-1, which enables invasion and egg products in the United States since the mid- of host cells, by the most recent common ancestor of 1990s due to multiple factors (120, 121). The recent all Salmonella subspecies, and SPI-2, for replication in emergence and spread of S. Heidelberg and S. Kentucky macrophages, during species divergence of S. enterica in poultry could be attributed to the acquisition of viru- from S. bongori (56). SPIs play a crucial role in the lence plasmids which harbor genes for iron acquisition, pathogenesis of S. enterica infections. So far, 24 SPIs colicin production, and disinfectant and heavy metal re- have been described and characterized. Of all SPIs sistance via HGT, providing a selective advantage in the reported in Salmonella, only SPI-1, SPI-4, SPI-5, and avian environment (107, 122, 123). Additionally, S. SPI-9 were acquired by Salmonella prior to speciation Enteritidis and S. Heidelberg share a common immuno- (56). The acquisition of SPI by HGT confers rapid dominant surface O antigen (O12) (124). The prevalence gain of complex virulence functions from other spe- of S. Heidelberg is partially due to the shared O12 anti- cies. Although several common motifs are present gen as well, as it competes for the same ecological niche among SPI, the distribution, size, structure, and func- with S. Enteritidis. Similar to S. Gallinarum and S. tion of these SPIs can be markedly different among Enteritidis, immunization of chickens specifically against subspecies, serovars, and/or strains. One such example infection with serovar S. Enteritidis (125, 126) led to the is SPI-3, which has at least four different versions and decrease of S. Enteritidis and expansion of serovar S. no identical copies within a single version (128). Other 10 EcoSalPlus.asm.org  Salmonella Genomics in Public Health and Food Safety examples are SPI-13 and SPI-8. SPI-13 is conserved in Rck and PagN, which allow salmonellae to invade host many serovars in clade A and clade B, except the cells (142). Comparative genomics revealed four gene Typhi clade (containing S. Typhi and S. Paratyphi A) clusters encoding T6SS, acquired by independent lateral in clade A, while SPI-8 is carried only by the Typhi transfer events, located in different genomic islands, clade at the same genomic location (56, 132, 133). including SPI-6, SPI-19, SPI-20, and SPI-21 (143). Different roles for SPI-13 and SPI-8 have been Interestingly, S. Enteritidis has a degenerate genetic ele- reported in intracellular macrophage survival (134). ment lacking about 22 T6SS-related open reading Moreover, several SPIs, such as SPI-7, are excisable frames (ORF) on SPI-19 with respect to S. Gallinarum, from Salmonella chromosome by site-specific recom- suggesting the role of T6SS in Salmonella evolution and bination, which can be of great epidemiological impor- host specificity. tance and may be a mechanism to regulate gene expression during human infection as well (135). Besides O and H antigens, Salmonella carries another group of surface proteins designated FGCs. Some types of Type III secretion systems (T3SS), encoded by SPI-1 and FGCs are restricted to a host, and some are carried by all SPI-2, respectively, play important roles in gastrointestinal Salmonella serovars, suggesting a potential role for fim- disease and systemic infection. Comparative genomic briae in host specificity. Comparison of 90 genomes and analysis reveals that all serovars of S. enterica encode a 60 plasmids of Salmonella revealed a fimbriome consisting subset of core effectors, and additional effectors are spor- of 35 different FGCs in the Salmonella pan-genome, each adically distributed among different serovars (132, 136), carrying the structural subunits and biogenesis genes of a suggesting that they are critical for virulence in different fimbria (90). The Salmonella fimbriome was extremely hosts. In addition, a third T3SS is responsible for the flag- diverse due to the extensive FGC deletion and acquisition ellum-based motility of the pathogen (56). Salmonella through HGT and to a high level of allelic variation in expresses a characteristic intracellular transcriptomic sig- predicted or known adhesins which parallel Salmonella nature in different cell types. Simultaneous expression of evolution toward host range modulation, survival, and three T3SSs suggested a time-dependent transcriptional persistence in specific niches, as well as strain virulence adaptation to the environment (137). The fluctuations in (90, 144). expression of mgtBC, pstACS, and iro genes, for magne- sium, phosphate, and iron uptake, and T3SS could reflect bacterial response to host cells during infection (137). Salmonella genomic evolution in the environment. Regulations of these virulence factors can occur both glob- Salmonellae possess multiple traits that permit survival ally and locally, forming complex feedback and feed-for- in a diverse set of environments, such as soils, sedi- ward regulatory loops. For example, transcription of hilA, ments, waters, and plant surfaces (145). In order to encoding the activator of the T3SS-1 structural genes, is endure in these environments, Salmonella must be able activated by three AraC-like proteins, HilD, HilC, and to overcome several stresses, including extremes of tem- RtsA, which each bind the promoter of hilA to directly perature, pH, salt/osmotic pressure, moisture, exposure enhance transcription (138, 139). to UV, and predators, to name a few (145, 146). Long- term persistence of these pathogens in the environment Many environmental signals, including low oxygen and has been documented. For example, Salmonella intro- high osmolarity, and regulatory systems such as the duced into corn crop soil through naturally contami- small RNAs (sRNAs) FnrS and AcrZ are integrated into nated poultry litter was found a year later (147). In this circuit to precisely regulate SPI-1 expression (140). surface waters from the Eastern Shore of Virginia, By base pairing with target mRNA or protein, sRNAs Salmonella with the same pulsed-field gel electrophoresis modulate expression of distinct regulons and key tran- (PFGE) pattern was isolated over multiple years (4, 148). scription factors and play an important role in major Some mechanisms that Salmonella uses for survival in stress response and virulence networks in Salmonella the environment are similar to those used during an (141). Moreover, recent advances in Salmonella patho- infection (145). Interestingly even some virulence factors genicity showed that Salmonella can cause infection in have been shown to be important to environmental sur- a T3SS-1-independent manner, which is mediated by vival. For example, Maserati et al. (149) demonstrated a large outer membrane proteins called invasins, namely, role for sopD and sseD in desiccation tolerance and EcoSalPlus.asm.org 11 Brown et al. survival. As more naturally occurring isolates are recov- In addition, nearly four dozen phages were identified ered and sequenced from various environments, more among the Salmonella isolates examined, with over unique adaptations for survival in those environments three quarters of isolates having an associated phage. may be discovered. Numerous isolates with multiple phages were observed often, and one isolate had up to 6 intact phages. Gifsy-1 and Fels-2 phages were commonly observed, with the Genomic evolution and phylogeny of Salmonella. Gifsy-2 phage also being common for this largely North FDA phylogenetic methods of evaluating Salmonella American and Asian sample set. Plasmid replicons are phylogeny enhance vertical evolution. The Center for commonly found in Salmonella isolates, with IncFI and Food Safety and Applied Nutrition (CFSAN) SNP pipe- IncFII regularly being present among many genomes of line is designed and validated to cluster isolates and to the more than two dozen other plasmid replicons iden- look for closely related shared ancestry (150). The HGT tified. Virulence factors (154–156) from type III secre- elements are examined in addition to the vertical signal, tion system (T3SS) SPI-1, including sipA, sipB, sipC, and with the vertical elements defining the phylogenetic sptP, were identified in all Salmonella genomes exam- clusters and the HGT elements defining any pathoge- ined, as were the SPI-2 T3SS genes spiC and ssaB. Other nicity, virulence, or AMR genes present among the iso- Salmonella virulence factors varied in clade presence or lates of interest. absence, suggesting a complex evolutionary pattern. Salmonella enterica is represented by.2,600 serovars, CRISPR-Cas systems have been identified in numerous making it difficult to fully place in a phylogenetic con- Salmonella genomes, with the alignments revealing mixed text with a single analysis and resultant phylogeny. homology across serovars with an increase of shared Investigators have attempted to capture all of the spacers toward the ancestral end of the CRISPR array. known diversity by using MLST (PubMLST [https:// Spacer alignments have revealed degradation of many in- pubmlst.org/salmonella/]) (151), and several MLST ternal spacers (57). The median number of spacers in schemes are available or have been proposed (34, 152, CRISPR 1 and 2 are 13 and 14, respectively, with the larg- 153). Large phylogenies have been built and diversity est array of 113 spacers being reported for Salmonella within the genus Salmonella has been described using enterica serovar Mbandaka. more of the WGS available data. Worley et al. (132) combined 445 isolate genomes from 266 distinct sero- Applications from long-read sequencing. There are vars and from 52 countries to build a comprehensive several different sequencing technologies that produce WGS phylogeny. An important finding from the study longer reads. Read lengths of 10,000 to 100,000 bp and was that more than 10% of the examined serovars, longer have been described. Once these longer reads nearly three dozen, designated by SeroSeq (24) were ei- exceeded roughly 11,000 bp, more Salmonella genomes ther polyphyletic or paraphyletic. These results suggest could be more easily and completely sequenced, having that the serovar markers have moved across the genus spanned a common major repeat. These closed genomes horizontally, though a clear timeline has not been estab- included the plasmids and phages associated with the lished or proposed. This WGS study reported on two bacterial genome. A closed genome refers to sequences previously unidentified S. enterica subsp. enterica clades that produce a single contig for each chromosome and labeled C and D, to add to the two other major lineages, mobile element present in the isolate. Numerous groups A and B, that other Salmonella phylogenetics works with access to these sequencing technologies began clos- have identified. Gifsy-1- and Gifsy-2-like phages appear ing genomes and plasmids with a focus on fully describ- more prevalent in clade A. Most virulence genes are ing the synteny of the genes on the chromosome and widely distributed across S. enterica, suggesting exten- plasmids. Knowing the specific order and presence of sive, frequent HGT and a more dynamic hypothesis for genes allowed investigators to determine new AMR bacterial evolution of the species. For antigen evolution, genes and the pathogenicity and virulence genes associ- observations suggest that the genes responsible for O ated with each unique isolate and plasmid. For most groups and phase 1 flagellar antigen traits are not evolv- foodborne-pathogen genomes, the presence of a known ing in a vertical fashion, suggesting an HGT role. AMR gene conferred the phenotype of resistance. 12 EcoSalPlus.asm.org  Salmonella Genomics in Public Health and Food Safety Sequences with multiple contigs may interrupt some food sample to the determination of the pathogen genes near long repetitive regions, confounding their genome sequences can take up to 10 to 12 days. identification. Genes may appear to be absent because Unlike clinical samples, food samples often con- they are only partially sequenced. The value of having a tain very low levels of Salmonella cells. In addi- fully closed and characterized genome allowed investiga- tion, the presence of competitive or antagonist tors to see all the genes that are present with more cer- organisms against salmonellae in the food micro- tainty, so that they can match gene presence with biome can pose serious challenges for effective phenotype presence. This also allowed discovery of new culture enrichment. Metagenomics, the collection AMR genes when the gene was absent but the phenotype of genomes and genes from the members of a was present and the discovery of allelic variants when the microbiota obtained through shotgun sequencing gene was present but the phenotype was absent. This of DNA extracted from a sample (159), however, strategy of closing genomes to better characterize is now beginning to provide a path forward in the Salmonella will work as well for any other gene that use of WGS technology for Salmonella detection investigators wish to characterize phenotypically, such as in situ in food and environmental backgrounds those involved in resistance to disinfectants and resist- (160). ance to desiccation or heat (157). Long-read sequencers have various levels of error, so some reads require polish- Despite the promise of metagenomic sequencing becoming ing with a higher-accuracy sequencing chemistry if the a one-stop solution in food microbiology laboratories, it still application being used is MLST or SNP-based phyloge- has several challenges to overcome. One of the greatest netics (158). Even long reads with higher error rates can challenges is the sensitivity and specificity of the current be useful, as the data can provide accurate scaffolds to metagenomic methods for direct detection of low levels of determine gene synteny and can be fully applied to pathogen of interest from high-background food micro- BLAST searches that discover the presence and absence biomes. Additionally, the choice of the extraction and of genes and plasmids against reference databases. An sequencing protocols and the type of controls and analysis additional benefit is the ability to sequence in the field using metagenomic software tools are other major chal- due to the mobile rapid nature of some of these tools. lenges to implementing and standardizing metagenomics for routine use in food microbiology laboratories (161, 162). Recent studies using a hybrid “quasi-metagenomic” Application of metagenomics in pathogen detection in food. A typical Salmonella isolation and identifica- approach demonstrated detection and subtyping of Shiga tion take 5 to 7 days using the isolate-centric work- toxin-producing Escherichia coli (STEC) from spinach flows described in the United States Food and Drug (163), Listeria monocytogenes in ice cream (164), and Administration’s bacteriological analytical manual (BAM) Salmonella enterica on cilantro (165), black peppercorn, (https://www.fda.gov/food/foodscienceresearch/ peanut butter, and lettuce (166) and in wheat flour (167). laboratorymethods/ucm2006949.htm) and the U.S. Quasi-metagenomic sequencing is a direct sequencing of Department of Agriculture Food Safety and Inspection microbiological enrichments (the first step in culture-based Service’s microbiology laboratory guidebook (MLG) detection methods). Sequencing of the modified micro- (https://www.fsis.usda.gov/wps/portal/fsis/topics/science/ biomes of food and environmental samples can provide laboratories-and-61procedures/guidebooks-and-methods/ high-resolution sequencing data for foodborne pathogen microbiology-laboratory-guidebook/microbiology detection and subtyping, expediting source tracking by up -62laboratory-guidebook). Further characteriza- to 4 to 6 days (168–170). These studies also underscore the tion of the isolates can increase the laboratory value of metagenomics as a tool to evaluate and rationalize turnaround time even more. The routine use of culture enrichment methods. Metagenomics reveals which WGS substantially reduces time and cost for pub- species grow along the enrichment timeline and documents lic health laboratories. However, current standard changes in species composition with various perturbations laboratory procedures for WGS, from regrowth of to enrichment protocols. Long-read sequencers also show the pathogen to actual sequencing, take up to promise for metagenomics methods, with the longer reads 5 days to complete. That means that the entire more accurately characterizing the species present in a process from the collection of a contaminated microbiome (171). EcoSalPlus.asm.org 13 Brown et al. Integration of genomics, investigation, and epidemiology. creates any problems, and in fact, the reverse is always Foodborne contamination events and outbreaks are the case, in that having more data provides higher reso- investigated by numerous federal and state partners, lution, which more clearly defines a contamination case including the FDA, USDA Food Safety and Inspection and the explicit genetic changes that have occurred Service (FSIS), CDC, and NCBI. Investigations are among the isolates sequenced (116). As more experi- supported by three lines of potential evidence. The ence is gained using WGS, the examples and evi- first lines of evidence often come from the laboratory, dence continue to show the powerful predictive role where WGS provides genetic support for a phyloge- that genomics plays in investigating contamination netic cluster that links food, environmental, and clini- events (11, 62, 83, 100, 108). Methods of reduced re- cal isolates. By focusing on the most closely related solution generally increase false inclusions, which isolates at the tips of the phylogenetic tree, WGS clus- are particularly problematic for ecological and epi- ters a subset of the isolates that are monophyletic and demiological models when clinical, food, and envi- share an ancestor. These subclusters are often used to ronmental isolates are included that were not part of separate outbreak signals from background noise, to the same contamination event. False inclusions mis- unravel the complexities of foodborne contaminations, direct investigations and reduce the power of pre- to support and prioritize epidemiological data, and to diction, ultimately delaying removal of the contaminant carry out site investigations. WGS unravels the com- from the food supply. plexity of a polyclonal outbreak by breaking the inves- tigation into smaller solvable parts. Each lineage High-resolution SNP analysis resolves all isolates within a polyclonal outbreak or contamination event down to the very tips of the tree (150). Phylogenetic is treated as an independent pathogen and piece of evi- trees are hierarchical, showing greater and greater re- dence tying a specific food commodity or firm to a solution from the base to the tip of the tree. If expo- clinical case. Epidemiological evidence may determine sure data suggest a common contaminant and or food whether the patients with clinical cases have been vehicle at a particular node that is supported by WGS exposed to a common contaminant found at a firm. data, then that node on the phylogenetic tree can be The FDA inspection may provide positive cultures of set as the case definition and scope of the outbreak. the foodborne pathogens contaminating the facility. Often there is clear evidence for a cluster break based The FDA relies on field inspectors to recover the di- on the number and/or positions of SNPs that define a versity of pathogens present in a contaminated facility. lineage and the bootstrap scores for the node. WGS For FDA compliance, it is often the inspection results provides additional evidence about the amount of genetic that determine whether a contamination event is poly- diversity that has accrued during a contamination event. It clonal. Also, multiple WGS clusters may each inde- is the high-resolution WGS data, combined with detailed pendently point back to the same firm being and structured metadata, that may be used by artificial intel- responsible for the contaminant exposure. The power ligence (AI) and machine learning (ML) tools to make even and prediction of the full investigation comes from more predictive models for the accurate prediction of food, integrating the various relevant pieces of evidence, animal source (172), and or geographic location. Published including those from laboratory and epidemiological WGS data have shown that most Salmonella and Listeria investigation. isolates exhibit a very strong phylogeographic signal that is highly predictive (173), based on the ability to predict with Genomic methods are always superior to lower-resolu- high probability whether a pathogen comes from the same tion subtyping methods when the goals are source facility, for isolates acquired during inspection. We also tracking, root cause investigation, and infectious disease know that isolates from clinical sources show similar levels control. Having more data is better for numerous rea- of genetic variability, suggesting that they would show simi- sons. The superior performance of WGS methods is the lar probabilities if comparable evidence was available to pre- reason why states and federal agencies have adopted dict the sources of their illness. WGS for all investigations of foodborne illness (12). WGS is best suited to integrate all case information, Risk assessment and risk management predictions also provided that its use is not delayed. We have not seen benefit from WGS data (174). FDA and GenomeTrakr any WGS evidence to suggest that having more data partners are including more detailed structured metadata 14 EcoSalPlus.asm.org  Salmonella Genomics in Public Health and Food Safety food ontology (175) (FoodOn, GenEpiOn, MixS, and case investigations, survey, surveillance, monitoring, IFSAC) to support efforts to foster innovation in AI and and outbreak investigations. The ISO creates standards ML. We have already seen numerous WGS examples of to facilitate trade by forming trust that is based on con- the power to predict country of origin (11), growing sensus among groups of experts in government, indus- region (176, 177), and even implicated egg farms (178). try, and academia. There are more than 21,000 ISO As we see phylogeographic structure in most of the trees we standards that address a wide variety of topics, includ- build, it is likely that AI and ML will contribute additional ing food microbiology. These standards develop trust future predictions to support contamination and outbreak among trading partners by standardizing the activities investigations. FDA investigators currently watch approxi- in which they are involved. For example, ISO develops mately 4,000 of the more than 40,000 clusters at the NCBI consensus positions on food microbiological standard meth- Pathogen Detection web site for isolates that cluster with ods in ISO Technical Committee (TC) 34/Subcommittee FDA foodborne pathogen genomes. This includes data (SC) 9. TC 34 is devoted to foods, and SC 9 is devoted to from roughly 340,000 Salmonella genomes, a number which microbiology. Within TC 34/SC 9, there is working group has grown from less than 1,000 in 2012. (WG) 25, “Whole-genome sequencing for typing and genomic characterization.” Cladistic methodology is one approach used to build phylo- genetic trees using parsimony methods. Cladistics is The WG 25 recently completed a committee draft uniquely valuable in optimizing characters on a phylogenetic (23418; “Whole-genome sequencing for typing and tree to predict when character variation occurs. Using these genomic characterization of foodborne bacteria—gen- methods, investigators can predict the unique changes that eral requirements and guidance”). The purpose of this define a lineage. The nucleotide changes that modify the standard is to address both the laboratory and bioin- coded amino acid (nonsynonymous changes) may also formatic components of WGS for foodborne microor- modify the protein and affect the phenotype. By combining ganisms. The overall goal of this standard is to provide cladistics, character optimization, and WGS, investigators consistency in the approach to WGS regardless of the may be able to identify genotype-to-phenotype changes that sequencing instrument, so that sequencing results will specific bacterial lineages have acquired and that allow food- be comparable throughout the world. The standard is borne pathogens to survive and contaminate foods, animals, in three parts: laboratory operations, validation, and and the environment (177). In several examples, investiga- metadata. Within these areas, the standard covers han- tors have predicted which genomic changes correlate with dling of bacterial cultures; genomic DNA isolation; outbreaks in Italian-style meats (178) and in eggs (115, 116, sequencing library preparation, sequencing, and 179), with the underlying phenotype predictions uncovering assessment of raw DNA sequence read quality and known pathogenicity and virulence gene variants and/or the storage; bioinformatics analysis for determining ability to infect the chicken host. These general methods will genetic relatedness, genetic content and predicting continue to be valuable for constructing genotype-to-pheno- phenotype, and bioinformatics pipeline validation; type hypotheses. metadata capture and sequence repository deposition; and validation of the end-to-end WGS workflow. These parameters are the minimum necessary for gen- Global genomic standards. Harmonization of test erating and analyzing WGS data obtained from food- protocols from different organizations, e.g., FDA, borne bacteria. International Organization for Standardization (ISO), AOAC International, and Association Française de ACKNOWLEDGMENTS Normalisation (AFNOR), has been pursued recently to We thank our partners at the United States Food and Drug facilitate global data sharing and comparison when Administration. We thank the many collaborators in the GenomeTrakr dealing with worldwide public health problems (180, network, CDC PulseNet, USDA-FSIS, who provide their data for broad 181). The need to validate newly developed or alterna- use and applications, and we acknowledge the data support that we all tive methods in comparison with established and stand- receive at the FDA and the NCBI. ard protocols, such as FDA, USDA, AOAC, ISO, and Work was funded by internal research funding from the U.S. AFNOR methods, has become urgent in recent years, in Food and Drug Administration. order to make sure that proper methods are used in all We declare no conflicts of interest, financial or otherwise. EcoSalPlus.asm.org 15 Brown et al. REFERENCES 16. Diep B, Barretto C, Portmann AC, Fournier C, Karczmarek A, Voets G, Li S, Deng X, Klijn A. 2019. Salmonella serotyping; 1. Grimont PA, Weill F-X. 2007. Antigenic formulae of the Salmo- comparison of the traditional method to a microarray-based method nella serovars, p 166. WHO Collaborating Centre for Reference and and an in silico platform using whole genome sequencing data. Front Research on Salmonella, Paris, France. 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