Publications by authors named "Marc W Allard"

114 Publications

Salmonella Genomics in Public Health and Food Safety.

EcoSal Plus 2021 Jun 14:eESP00082020. Epub 2021 Jun 14.

Center for Food Safety and Applied Nutrition, U.S. Food and Drug Administration, College Park, Maryland, USA.

The species Salmonella enterica comprises over 2,600 serovars, 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 environmental niches, including water, soil, and various plant (including produce) species. Much of what is known about the evolution and diversity of nontyphoidal Salmonella serovars (NTS) in the environment is the result of the rise of the genomics era in enteric microbiology. There are over 340,000 Salmonella genomes available in public databases. This extraordinary breadth of genomic diversity now available for the species, coupled with widespread availability and affordability of whole-genome sequencing (WGS) instrumentation, has transformed the way in which we detect, differentiate, and characterize Salmonella enterica strains in a timely way. Not only have WGS data afforded a detailed and global examination of the molecular epidemiological movement of Salmonella from diverse environmental reservoirs into human and animal hosts, but they have also allowed considerable consolidation of the diagnostic effort required to test for various phenotypes important to the characterization of Salmonella. For example, drug resistance, serovar, virulence determinants, and other genome-based attributes can all be discerned using a genome sequence. Finally, genomic analysis, in conjunction with functional and phenotypic approaches, is beginning to provide new insights into the precise adaptive changes that permit persistence of NTS in so many diverse and challenging environmental niches.
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http://dx.doi.org/10.1128/ecosalplus.ESP-0008-2020DOI Listing
June 2021

Genomic surveillance of antimicrobial resistance shows cattle and poultry are a moderate source of multi-drug resistant non-typhoidal Salmonella in Mexico.

PLoS One 2021 5;16(5):e0243681. Epub 2021 May 5.

Facultad de Medicina Veterinaria y Zootecnia, Universidad Nacional Autónoma de México, Ciudad de México, México.

Multi-drug resistant (MDR) non-typhoidal Salmonella (NTS) is a public health concern globally. This study reports the phenotypic and genotypic antimicrobial resistance (AMR) profiles of NTS isolates from bovine lymph nodes (n = 48) and ground beef (n = 29). Furthermore, we compared genotypic AMR data of our isolates with those of publicly available NTS genomes from Mexico (n = 2400). The probability of finding MDR isolates was higher in ground beef than in lymph nodes:χ2 = 12.0, P = 0.0005. The most common resistant phenotypes involved tetracycline (40.3%), carbenicillin (26.0%), amoxicillin-clavulanic acid (20.8%), chloramphenicol (19.5%) and trimethoprim-sulfamethoxazole (16.9%), while more than 55% of the isolates showed decreased susceptibility to ciprofloxacin and 26% were MDR. Conversely, resistance to cephalosporins and carbapenems was infrequent (0-9%). MDR phenotypes were strongly associated with NTS serovar (χ2 = 24.5, P<0.0001), with Typhimurium accounting for 40% of MDR strains. Most of these (9/10), carried Salmonella genomic island 1, which harbors a class-1 integron with multiple AMR genes (aadA2, blaCARB-2, floR, sul1, tetG) that confer a penta-resistant phenotype. MDR phenotypes were also associated with mutations in the ramR gene (χ2 = 17.7, P<0.0001). Among public NTS isolates from Mexico, those from cattle and poultry had the highest proportion of MDR genotypes. Our results suggest that attaining significant improvements in AMR meat safety requires the identification and removal (or treatment) of product harboring MDR NTS, instead of screening for Salmonella spp. or for isolates showing resistance to individual antibiotics. In that sense, massive integration of whole genome sequencing (WGS) technologies in AMR surveillance provides the shortest path to accomplish these goals.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0243681PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8099073PMC
May 2021

Insights about the epidemiology of Salmonella Typhimurium isolates from different sources in Brazil using comparative genomics.

Gut Pathog 2021 Apr 28;13(1):27. Epub 2021 Apr 28.

Departamento de Análises Clínicas, Toxicológicas e Bromatológicas, Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo - USP, Ribeirão Preto, Brazil.

Background: Salmonella enterica subsp. enterica serovar Typhimurium (S. Typhimurium) is an important zoonotic agent worldwide. The aim of this work was to compare genetically 117 S. Typhimurium isolated from different sources over 30 years in Brazil using different genomics strategies.

Results: The majority of the 117 S. Typhimurium strains studied were grouped into a single cluster (≅ 90%) by the core genome multilocus sequence typing and (≅ 77%) by single copy marker genes. The phylogenetic analysis based on single nucleotide polymorphism (SNP) grouped most strains from humans into a single cluster (≅ 93%), while the strains isolated from food and swine were alocated into three clusters. The different orthologous protein clusters found for some S. Typhimurium isolated from humans and food are involved in metabolic and regulatory processes. For 26 isolates from swine the sequence types (ST) 19 and ST1921 were the most prevalent ones, and the ST14, ST64, ST516 and ST639 were also detected. Previous results typed the 91 S. Typhimurium isolates from humans and foods as ST19, ST313, ST1921, ST3343 and ST1649. The main prophages detected were: Gifsy-2 in 79 (67.5%) and Gifsy-1 in 63 (54%) strains. All of the S. Typhimurium isolates contained the acrA, acrB, macA, macB, mdtK, emrA, emrB, emrR and tolC efflux pump genes.

Conclusions: The phylogenetic trees grouped the majority of the S. Typhimurium isolates from humans into a single cluster suggesting that there is one prevalent subtype in Brazil. Regarding strains isolated from food and swine, the SNPs' results suggested the circulation of more than one subtype over 30 years in this country. The orthologous protein clusters analysis revealed unique genes in the strains studied mainly related to bacterial metabolism. S. Typhimurium strains from swine showed greater diversity of STs and prophages in comparison to strains isolated from humans and foods. The pathogenic potential of S. Typhimurium strains was corroborated by the presence of exclusive prophages of this serovar involved in its virulence. The high number of resistance genes related to efflux pumps is worrying and may lead to therapeutic failures when clinical treatment is needed.
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http://dx.doi.org/10.1186/s13099-021-00423-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8082823PMC
April 2021

Enhanced biofilm and extracellular matrix production by chronic carriage versus acute isolates of Salmonella Typhi.

PLoS Pathog 2021 01 19;17(1):e1009209. Epub 2021 Jan 19.

Center for Microbial Pathogenesis, Research Institute at Nationwide Children's Hospital, Columbus, Ohio, United States of America.

Salmonella Typhi is the primary causative agent of typhoid fever; an acute systemic infection that leads to chronic carriage in 3-5% of individuals. Chronic carriers are asymptomatic, difficult to treat and serve as reservoirs for typhoid outbreaks. Understanding the factors that contribute to chronic carriage is key to development of novel therapies to effectively resolve typhoid fever. Herein, although we observed no distinct clustering of chronic carriage isolates via phylogenetic analysis, we demonstrated that chronic isolates were phenotypically distinct from acute infection isolates. Chronic carriage isolates formed significantly thicker biofilms with greater biomass that correlated with significantly higher relative levels of extracellular DNA (eDNA) and DNABII proteins than biofilms formed by acute infection isolates. Importantly, extracellular DNABII proteins include integration host factor (IHF) and histone-like protein (HU) that are critical to the structural integrity of bacterial biofilms. In this study, we demonstrated that the biofilm formed by a chronic carriage isolate in vitro, was susceptible to disruption by a specific antibody against DNABII proteins, a successful first step in the development of a therapeutic to resolve chronic carriage.
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http://dx.doi.org/10.1371/journal.ppat.1009209DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7815147PMC
January 2021

Preserve a Voucher Specimen! The Critical Need for Integrating Natural History Collections in Infectious Disease Studies.

mBio 2021 01 12;12(1). Epub 2021 Jan 12.

University of Vermont, Burlington, Vermont, USA.

Despite being nearly 10 months into the COVID-19 (coronavirus disease 2019) pandemic, the definitive animal host for SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2), the causal agent of COVID-19, remains unknown. Unfortunately, similar problems exist for other betacoronaviruses, and no vouchered specimens exist to corroborate host species identification for most of these pathogens. This most basic information is critical to the full understanding and mitigation of emerging zoonotic diseases. To overcome this hurdle, we recommend that host-pathogen researchers adopt vouchering practices and collaborate with natural history collections to permanently archive microbiological samples and host specimens. Vouchered specimens and associated samples provide both repeatability and extension to host-pathogen studies, and using them mobilizes a large workforce (i.e., biodiversity scientists) to assist in pandemic preparedness. We review several well-known examples that successfully integrate host-pathogen research with natural history collections (e.g., yellow fever, hantaviruses, helminths). However, vouchering remains an underutilized practice in such studies. Using an online survey, we assessed vouchering practices used by microbiologists (e.g., bacteriologists, parasitologists, virologists) in host-pathogen research. A much greater number of respondents permanently archive microbiological samples than archive host specimens, and less than half of respondents voucher host specimens from which microbiological samples were lethally collected. To foster collaborations between microbiologists and natural history collections, we provide recommendations for integrating vouchering techniques and archiving of microbiological samples into host-pathogen studies. This integrative approach exemplifies the premise underlying One Health initiatives, providing critical infrastructure for addressing related issues ranging from public health to global climate change and the biodiversity crisis.
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http://dx.doi.org/10.1128/mBio.02698-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7844540PMC
January 2021

Antimicrobial resistance and related gene analysis of Salmonella from egg and chicken sources by whole-genome sequencing.

Poult Sci 2020 Dec 12;99(12):7076-7083. Epub 2020 Oct 12.

Division of Microbiology, Office of Regulatory Science, Center for Food Safety and Nutrition, U.S. Food and Drug Administration, College Park, MD. Electronic address:

Whole-genome sequencing (WGS) is a valuable tool in research on foodborne pathogens. In this study, a total of 143 isolates of Salmonella serotypes Enteritidis, Typhimurium, and Heidelberg sourced from eggs and chickens were analyzed for their antimicrobial resistance profiles using WGS data. The isolates carried high rate of genes resistant to aminoglycoside (70.63%), tetracycline (26.57%), fosfomycin (25.17%), sulfonamides (23.78%), and β-lactamases (15.38%); and aadA was the most frequently observed antimicrobial resistance gene (ARG). Antimicrobial resistance varies by Salmonella serotypes, with Salmonella enterica serovar Enteritidis (Salmonella ser. Enteritidis) isolates being highly resistant to aminoglycoside (particularly streptomycin); Salmonella ser. Typhimurium more resistant to aminoglycoside, tetracycline, and sulfonamides; and Salmonella ser. Heidelberg more resistant to aminoglycoside and fosfomycin. Salmonella ser. Typhimurium isolates presented more varieties of ARG than Salmonella ser. Enteritidis and Salmonella ser. Heidelberg. Our data showed that 5 isolates of Salmonella ser. Typhimurium and Salmonella ser. Heidelberg contained ARG resistant to ≥ 5 antimicrobials. In addition, 23 Salmonella isolates carried ARG resistant to 4 antimicrobials.
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http://dx.doi.org/10.1016/j.psj.2020.10.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7705029PMC
December 2020

Food Safety Genomics and Connections to One Health and the Clinical Microbiology Laboratory.

Clin Lab Med 2020 12 5;40(4):553-563. Epub 2020 Oct 5.

US Food and Drug Administration, Center for Food Safety and Applied Nutrition, 5001 Campus Drive, College Park, MD 20740, USA.

This article describes the potential for one health surveillance of foodborne pathogens and disease using the revolutionary methodologies of whole genome sequencing. Whole genome sequencing of viral and bacterial pathogens is a natural fit to a one health perspective because these pathogens reside and are shared by humans, animals, and the environment and their genomes are compared easily regardless of where or from what host the pathogen was isolated. A genome provides a huge amount of data that can be analyzed for numerous applications. Sharing data coordinates surveillance efforts across the various disciplines.
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http://dx.doi.org/10.1016/j.cll.2020.08.011DOI Listing
December 2020

Phenotypic and genotypic characterization of Salmonella Typhimurium isolates from humans and foods in Brazil.

PLoS One 2020 18;15(8):e0237886. Epub 2020 Aug 18.

Departamento de Análises Clínicas, Toxicológicas e Bromatológicas, Faculdade de Ciências Farmacêuticas de Ribeirão Preto-Universidade de São Paulo-USP, Brazil.

Salmonella enterica subsp. enterica serovar Typhimurium (S. Typhimurium) causes gastroenteritis in many countries. However, in Brazil there are few studies that have conducted a virulence characterization of this serovar. The aim of this study was to evaluate the virulence potential of S. Typhimurium strains isolated in Brazil. Forty S. Typhimurium strains isolated from humans (n = 20) and food (n = 20) from Brazil were studied regarding their invasion and survival in human epithelial cells (Caco-2) and macrophages (U937). Their virulence potential was determined using the Galleria mellonella larvae model combined with the analysis of virulence genes by whole genome sequencing (WGS). A total of 67.5% of the S. Typhimurium studied (32.5% isolated from humans and 35% isolated from food) invaded Caco-2 epithelial cells at levels similar to or greater than the S. Typhimurium SL1344 prototype strain. In addition, 37.5% of the studied strains (25% isolated from humans and 12.5% isolated from food) survived in U937 human macrophages at levels similar to or greater than SL1344. S. Typhimurium strains isolated from humans (40%) and food (25%) showed high or intermediate virulence in G. mellonella larvae after seven days exposure. Approximately, 153 virulence genes of chromosomal and plasmidial origin were detected in the strains studied. In conclusion, the ability of the S. Typhimurium to invade Caco-2 epithelial cells was strain dependent and was not related to the source or the year of isolation. However, S. Typhimurium strains isolated from humans showed greater survival rates in U937 human macrophages, and presented higher proportion of isolates with a virulent profile in G. mellonella in comparison to strains isolated from food suggesting that this difference may be related to the higher frequency of human isolates which contained plasmid genes, such as spvABCDR operon, pefABCD operon, rck and mig-5.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0237886PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7437471PMC
October 2020

Microevolution and Gain or Loss of Mobile Genetic Elements of Outbreak-Related in Food Processing Environments Identified by Whole Genome Sequencing Analysis.

Front Microbiol 2020 29;11:866. Epub 2020 May 29.

Center for Food Safety and Applied Nutrition, Food and Drug Administration, College Park, MD, United States.

Whole genome sequencing (WGS) analyses have been instrumental in traceback investigations of (). To demonstrate how long-read sequencing analysis can capture and describe relationships among isolates from clinical, food, and environmental sources, we analyzed 366 long-read- and shotgun-sequenced isolates from 16 outbreak strains associated with cantaloupe, leafy green, stone fruit, caramel apple, mung bean sprout, multiple cheese products, multiple ice cream products, and their production environments. The analyses demonstrated that outbreak strains could be distributed in different areas and zones of food production environments through persistent or repeated contamination. Multi-strain and multi-clone contamination were common. Further, WGS could differentiate among isolates collected at different time points or from different production lines in the same facility, revealing microevolution events in processing environments. Our comparison between complete and shotgun genomes showed that isolates of the same outbreak strain diversified mostly by gain/loss of plasmids and chromosome-borne prophages that constitute 2 to 5% of the chromosome. In contrast, other genes missing in the shotgun genomes were randomly scattered, constituting ~0.5% of the chromosome. Among different outbreak strains of the same CC, most gene-scale differences were due to gain/loss of mobile genetic elements, such as plasmids, chromosome-borne prophages, a Tn916 like transposon, and Genomic Island 2. The nucleotide variations in the same prophage and the same plasmid shared among isolates of the same outbreak strain were limited, which enabled different WGS tools to unambiguously cluster isolates of the same outbreak strain. In some outbreak strains, correlation between prophage gain/loss and single nucleotide polymorphism (SNP) accumulations in the genome backbone were observed.
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http://dx.doi.org/10.3389/fmicb.2020.00866DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7272582PMC
May 2020

Draft Genome Sequences of Two Extensively Drug-Resistant Strains of Acinetobacter baumannii Isolated from Clinical Samples in Pakistan.

Microbiol Resour Announc 2020 May 14;9(20). Epub 2020 May 14.

Division of Microbiology, Office of Regulatory Science, Center for Food Safety and Applied Nutrition, U.S. Food and Drug Administration, College Park, Maryland, USA.

Infections in immunocompromised patients that are caused by extensively drug-resistant (XDR) strains have been increasingly reported worldwide. In particular, carbapenem-resistant strains are a prominent cause of health care-associated infections. Here, we report draft genome assemblies for two clinical XDR isolates obtained from hospitalized patients in Pakistan.
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http://dx.doi.org/10.1128/MRA.00026-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7225525PMC
May 2020

Temporal Dynamics of subsp. Serovar Agona Isolates From a Recurrent Multistate Outbreak.

Front Microbiol 2020 23;11:478. Epub 2020 Mar 23.

Division of Public Health and Biostatistics, Office of Food Defense, Communication and Emergency Response, Center for Food Safety and Applied Nutrition, U.S. Food & Drug Administration, College Park, MD, United States.

The largest outbreak of Agona in the United States occurred in 1998. It affected more than 400 patients and was linked to toasted oat cereal. Ten years later, a similar outbreak occurred with the same outbreak strain linked to the same production facility. In this study, whole-genome sequence (WGS) data from a set of 46 Agona including five isolates associated with the 1998 outbreak and 25 isolates associated with the 2008 outbreak were analyzed. From each outbreak one isolate was sequenced on the Pacific Biosciences Sequencer to determine the complete genome sequence. We reconstructed a phylogenetic hypothesis of the samples using a reference-based method for identifying variable sites. Using Single Nucleotide Polymorphism (SNP) analyses, we were able to distinguish and separate Agona isolates from both outbreaks with only a mean of eight SNP differences between them. The phylogeny illustrates that the 2008 outbreak involves direct descendants from the 1998 outbreak rather than a second independent contamination event. Based on these results, there is evidence supporting the persistence of over time in food processing facilities and highlights the need for consistent monitoring and control of organisms in the supply chain to minimize the risk of successive outbreaks.
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http://dx.doi.org/10.3389/fmicb.2020.00478DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7104706PMC
March 2020

Attribution of Listeria monocytogenes human infections to food and animal sources in Northern Italy.

Food Microbiol 2020 Aug 20;89:103433. Epub 2020 Jan 20.

University of Turin. Largo P, Braccini, 2, 10095, Grugliasco, Italy; US Food & Drug Administration. 5001 Campus Drive, 20740, College Park, MD, USA. Electronic address:

Listeriosis is a foodborne illness characterized by a relatively low morbidity, but a large disease burden due to the severity of clinical manifestations and the high case fatality rate. Increased listeriosis notifications have been observed in Europe since the 2000s. However, the reasons for this increase are largely unknown, with the sources of sporadic human listerioris often remaining elusive. Here we inferred the relative contributions of several putative sources of Listeria monocytogenes strains from listerioris patients in Northern Italy (Piedmont and Lombardy regions), using two established source attribution models (i.e. 'Dutch' and 'STRUCTURE') in comparative fashion. We compared the Multi-Locus Sequence Typing and Multi-Virulence-Locus Sequence Typing profiles of strains collected from beef, dairy, fish, game, mixed foods, mixed meat, pork, and poultry. Overall, 634 L. monocytogenes isolates were collected from 2005 to 2016. In total, 40 clonal complexes and 51 virulence types were identified, with 36% of the isolates belonging to possible epidemic clones (i.e. genetically related strains from unrelated outbreaks). Source attribution analysis showed that 50% of human listerioris cases (95% Confidence Interval 44-55%) could be attributed to dairy products, followed by poultry and pork (15% each), and mixed foods (15%). Since the contamination of dairy, poultry and pork products are closely linked to primary production, expanding actions currently limited to ready-to-eat products to the reservoir level may help reducing the risk of cross-contamination at the consumer level.
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http://dx.doi.org/10.1016/j.fm.2020.103433DOI Listing
August 2020

Correction to: Utilizing the Public GenomeTrakr Database for Foodborne Pathogen Traceback.

Methods Mol Biol 2019 ;1918:C1

Center for Food Safety and Applied Nutrition, U.S. Food and Drug Administration, College Park, MD, USA.

The chapter "Utilizing the Public GenomeTrakr Database for Foodborne Pathogen Traceback" is changed to open access, per the author's request in this revised version of the book.
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http://dx.doi.org/10.1007/978-1-4939-9000-9_22DOI Listing
January 2019

Atypical Salmonella enterica Serovars in Murine and Human Macrophage Infection Models.

Infect Immun 2020 03 23;88(4). Epub 2020 Mar 23.

UCD Centre for Food Safety, School of Public Health, Physiotherapy and Sports Science, University College Dublin, Belfield, Dublin, Ireland

Nontyphoidal species are globally disseminated pathogens and are the predominant cause of gastroenteritis. The pathogenesis of salmonellosis has been extensively studied using murine models and cell lines, typically challenged with serovar Typhimurium. Although serovars Enteritidis and Typhimurium are responsible for most of the human infections reported to the Centers for Disease Control and Prevention (CDC), several other serovars also contribute to clinical cases of salmonellosis. Despite their epidemiological importance, little is known about their infection phenotypes. Here, we report the virulence characteristics and genomes of 10 atypical serovars linked to multistate foodborne outbreaks in the United States. We show that the murine RAW 264.7 macrophage model of infection is unsuitable for inferring human-relevant differences in nontyphoidal infections, whereas differentiated human THP-1 macrophages allowed these isolates to be further characterized in a more human-relevant context.
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http://dx.doi.org/10.1128/IAI.00353-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7093118PMC
March 2020

Phylogenetic analysis revealed that Salmonella Typhimurium ST313 isolated from humans and food in Brazil presented a high genomic similarity.

Braz J Microbiol 2020 Mar 15;51(1):53-64. Epub 2019 Nov 15.

Departamento de Análises Clínicas, Toxicológicas e Bromatológicas, Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo - USP, Av. do Café, s/n°-Campus Universitário USP, Ribeirão Preto, SP, 14040-903, Brazil.

Salmonella Typhimurium sequence type 313 (S. Typhimurium ST313) has caused invasive disease mainly in sub-Saharan Africa. In Brazil, ST313 strains have been recently described, and there is a lack of studies that assessed by whole genome sequencing (WGS)-the relationship of these strains. The aims of this work were to study the phylogenetic relationship of 70 S. Typhimurium genomes comparing strains of ST313 (n = 9) isolated from humans and food in Brazil among themselves, with other STs isolated in this country (n = 31) and in other parts of the globe (n = 30) by 16S rRNA sequences, the Gegenees software, whole genome multilocus sequence typing (wgMLST), and average nucleotide identity (ANI) for the genomes of ST313. Additionally, pangenome analysis was performed to verify the heterogeneity of these genomes. The phylogenetic analyses showed that the ST313 genomes were very similar among themselves. However, the ST313 genomes were usually clustered more distantly to other STs of strains isolated in Brazil and in other parts of the world. By pangenome calculation, the core genome was 2,880 CDSs and 4,171 CDSs singletons for all the 70 S. Typhimurium genomes studied. Considering the 10 ST313 genomes analyzed the core genome was 4,112 CDSs and 76 CDSs singletons. In conclusion, the ST313 genomes from Brazil showed a high similarity among them which information might eventually help in the development of vaccines and antibiotics. The pangenome analysis showed that the S. Typhimurium genomes studied presented an open pangenome, but specifically tending to become close for the ST313 strains.
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http://dx.doi.org/10.1007/s42770-019-00155-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7058764PMC
March 2020

Characterization of Isolates from Selected U.S. Swine Feed Mills by Whole-Genome Sequencing.

Foodborne Pathog Dis 2020 02 8;17(2):126-136. Epub 2019 Nov 8.

Center for Food Safety and Applied Nutrition, U.S. Food and Drug Administration, College Park, Maryland.

Every year salmonellosis is responsible for $2.3 billion in costs to the U.S. food industry, with nearly 6% of the reported cases associated with pork and/or pork products. Several studies have demonstrated the role of pigs as reservoirs. Furthermore, this pathogen has been identified as a potential biological hazard in many livestock feeds. The overall objective of this research was to characterize isolates in selected U.S. swine feed mills by whole-genome sequencing (WGS) and evaluate isolates in association with the season and feed production stages. isolates were collected from 11 facilities during a previous study. Samples were analyzed for prevalence following the U.S. Department of Agriculture guidelines and confirmed by PCR. WGS was carried out on either the MiSeq or NextSeq sequencer. genome assemblies were obtained with the Shovill pipeline, version 0.9. ResFinder and SPIFinder were used to identify antibiotic resistance genes and pathogenicity islands. Finally, their phylogenetic relationship and diversity were determined by core genome multilocus sequence typing. Overall, our analysis showed the presence of in the feed mill environment. Isolates belonged to 16 different serotypes. Agona, Mbandaka, Senfenberg, and Scharzengrund were the most frequently found, and 18 single-nucleotide polymorphism clusters were identified. analysis showed that 40% of the strains carried at least one antimicrobial resistance gene. All isolates in this study could be considered of public health concern and pathogenic potential. Our findings underscore the potential role of the feed mill environment as the pathogen entry route into the human food value chain.
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http://dx.doi.org/10.1089/fpd.2019.2701DOI Listing
February 2020

Draft Genome Sequences of Antimicrobial-Resistant Clinical Isolates from Pakistan.

Microbiol Resour Announc 2019 Jul 25;8(30). Epub 2019 Jul 25.

Division of Microbiology, Office of Regulatory Science, Center for Food Safety and Applied Nutrition, U.S. Food and Drug Administration, College Park, Maryland, USA.

spp. are the most common cause of dysentery in developing countries and the second leading cause of diarrheal deaths worldwide. Multidrug-resistant (MDR) spp. are a serious threat to global health. Herein, we report draft genome sequences for three MDR isolates from Pakistan, two isolates and one isolate.
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http://dx.doi.org/10.1128/MRA.00500-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6658682PMC
July 2019

All for one and one for all: the true potential of whole-genome sequencing.

Lancet Infect Dis 2019 07 24;19(7):683-684. Epub 2019 May 24.

US Food and Drug Administration, College Park, VA 20740, USA.

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http://dx.doi.org/10.1016/S1473-3099(19)30172-0DOI Listing
July 2019

Whole genome sequencing uses for foodborne contamination and compliance: Discovery of an emerging contamination event in an ice cream facility using whole genome sequencing.

Infect Genet Evol 2019 09 27;73:214-220. Epub 2019 Apr 27.

US Food and Drug Administration, 5001 Campus Drive, College Park, MD, USA.

We review how FDA surveillance identifies several ways that whole genome sequencing (WGS) improves actionable outcomes for public health and compliance in a case involving Listeria monocytogenes contamination in an ice cream facility. In late August 2017 FDA conducted environmental sampling inside an ice cream facility. These isolates were sequenced and deposited into the GenomeTrakr databases. In September 2018 the Centers for Disease Control and Prevention contacted the Florida Department of Health after finding that the pathogen analyses of three clinical cases of listeriosis (two in 2013, one in 2018) were highly related to the aforementioned L. monocytogenes isolates collected from the ice cream facility. in 2017. FDA returned to the ice cream facility in late September 2018 and conducted further environmental sampling and again recovered L. monocytogenes from environmental subsamples that were genetically related to the clinical cases. A voluntary recall was issued to include all ice cream manufactured from August 2017 to October 2018. Subsequently, FDA suspended this food facility's registration. WGS results for L. monocytogenes found in the facility and from clinical samples clustered together by 0-31 single nucleotide polymorphisms (SNPs). The FDA worked together with the Centers for Disease Control and Prevention, as well as the Florida Department of Health, and the Florida Department of Agriculture and Consumer Services to recall all ice cream products produced by this facility. Our data suggests that when available isolates from food facility inspections are subject to whole genome sequencing and the subsequent sequence data point to linkages between these strains and recent clinical isolates (i.e., <20 nucleotide differences), compliance officials should take regulatory actions early to prevent further potential illness. The utility of WGS for applications related to enforcement of FDA compliance programs in the context of foodborne pathogens is reviewed.
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http://dx.doi.org/10.1016/j.meegid.2019.04.026DOI Listing
September 2019

Phylogenomic Pipeline Validation for Foodborne Pathogen Disease Surveillance.

J Clin Microbiol 2019 05 26;57(5). Epub 2019 Apr 26.

Center for Food Safety and Applied Nutrition, U.S. Food and Drug Administration, College Park, Maryland, USA.

Foodborne pathogen surveillance in the United States is transitioning from strain identification using restriction digest technology (pulsed-field gel electrophoresis [PFGE]) to shotgun sequencing of the entire genome (whole-genome sequencing [WGS]). WGS requires a new suite of analysis tools, some of which have long histories in academia but are new to the field of public health and regulatory decision making. Although the general workflow is fairly standard for collecting and analyzing WGS data for disease surveillance, there are a number of differences in how the data are collected and analyzed across public health agencies, both nationally and internationally. This impedes collaborative public health efforts, so national and international efforts are underway to enable direct comparison of these different analysis methods. Ultimately, the harmonization efforts will allow the (mutually trusted and understood) production and analysis of WGS data by labs and agencies worldwide, thus improving outbreak response capabilities globally. This review provides a historical perspective on the use of WGS for pathogen tracking and summarizes the efforts underway to ensure the major steps in phylogenomic pipelines used for pathogen disease surveillance can be readily validated. The tools for doing this will ensure that the results produced are sound, reproducible, and comparable across different analytic approaches.
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http://dx.doi.org/10.1128/JCM.01816-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6498022PMC
May 2019

Genomic surveillance links livestock production with the emergence and spread of multi-drug resistant non-typhoidal Salmonella in Mexico.

J Microbiol 2019 Apr 5;57(4):271-280. Epub 2019 Feb 5.

Facultad de Medicina Veterinaria y Zootecnia, Universidad Nacional Autónoma de México, Mexico City, Mexico.

Multi-drug resistant (MDR) non-typhoidal Salmonella (NTS) is increasingly common worldwide. While food animals are thought to contribute to the growing antimicrobial resistance (AMR) problem, limited data is documenting this relationship, especially in low and middle-income countries (LMIC). Herein, we aimed to assess the role of non-clinical NTS of bovine origin as reservoirs of AMR genes of human clinical significance. We evaluated the phenotypic and genotypic AMR profiles in a set of 44 bovine-associated NTS. For comparative purposes, we also included genotypic AMR data of additional isolates from Mexico (n = 1,067) that are publicly available. The most frequent AMR phenotypes in our isolates involved tetracycline (40/44), trimethoprim-sulfamethoxazole (26/44), chloramphenicol (19/44), ampicillin (18/44), streptomycin (16/44), and carbenicillin (13/44), while nearly 70% of the strains were MDR. These phenotypes were correlated with a widespread distribution of AMR genes (i.e. tetA, aadA, dfrA12, dfrA17, sul1, sul2, bla-TEM-1, blaCARB-2) against multiple antibiotic classes, with some of them contributed by plasmids and/or class-1 integrons. We observed different AMR genotypes for betalactams and tetracycline resistance, providing evidence of convergent evolution and adaptive AMR. The probability of MDR genotype occurrence was higher in meat-associated isolates than in those from other sources (odds ratio 11.2, 95% confidence interval 4.5-27.9, P < 0.0001). The study shows that beef cattle are a significant source of MDR NTS in Mexico, highlighting the role of animal production on the emergence and spread of MDR Salmonella in LMIC.
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http://dx.doi.org/10.1007/s12275-019-8421-3DOI Listing
April 2019

The use of next generation sequencing for improving food safety: Translation into practice.

Food Microbiol 2019 Jun 17;79:96-115. Epub 2018 Nov 17.

Gastrointestinal Bacteria Reference Unit, National Infection Service, Public Health England, 61 Colindale Avenue, London, NW9 5EQ, United Kingdom. Electronic address:

Next Generation Sequencing (NGS) combined with powerful bioinformatic approaches are revolutionising food microbiology. Whole genome sequencing (WGS) of single isolates allows the most detailed comparison possible hitherto of individual strains. The two principle approaches for strain discrimination, single nucleotide polymorphism (SNP) analysis and genomic multi-locus sequence typing (MLST) are showing concordant results for phylogenetic clustering and are complementary to each other. Metabarcoding and metagenomics, applied to total DNA isolated from either food materials or the production environment, allows the identification of complete microbial populations. Metagenomics identifies the entire gene content and when coupled to transcriptomics or proteomics, allows the identification of functional capacity and biochemical activity of microbial populations. The focus of this review is on the recent use and future potential of NGS in food microbiology and on current challenges. Guidance is provided for new users, such as public health departments and the food industry, on the implementation of NGS and how to critically interpret results and place them in a broader context. The review aims to promote the broader application of NGS technologies within the food industry as well as highlight knowledge gaps and novel applications of NGS with the aim of driving future research and increasing food safety outputs from its wider use.
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http://dx.doi.org/10.1016/j.fm.2018.11.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6492263PMC
June 2019

Genome divergence and increased virulence of outbreak associated subspecies .

Gut Pathog 2018 24;10:53. Epub 2018 Dec 24.

1Department of Veterinary and Biomedical Sciences, South Dakota State University, Brookings, SD USA.

serotype is primarily a poultry adapted serotype of Salmonella that can also colonize other hosts and cause human disease. In this study, we compared the genomes of outbreak associated non-outbreak causing ser. strains from diverse hosts and geographical regions. Human outbreak associated strains in this study were from a 2015 multistate outbreak of ser. involving 15 states in the United States which originated from bull calves. Our clinicopathologic examination revealed that cases involving ser. strains were predominantly young, less than weeks-old, dairy calves. Pre-existing or concurrent disease was found in the majority of the calves. Detection of ser. correlated with markedly increased death losses clinically comparable to those seen in herds infected with Dublin, a known serious pathogen of cattle. Whole genome based single nucleotide polymorphism based analysis revealed that these calf isolates formed a distinct cluster along with outbreak associated human isolates. The defining feature of the outbreak associated strains, when compared to older isolates of . , is that all isolates in this cluster contained fimbrial genes which are generally absent in . . The acquisition of several single nucleotide polymorphisms and the gain of fimbrial genes may have contributed to the increased disease severity of these ser. strains.
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http://dx.doi.org/10.1186/s13099-018-0279-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6304783PMC
December 2018

Utilizing the Public GenomeTrakr Database for Foodborne Pathogen Traceback.

Methods Mol Biol 2019 ;1918:201-212

Center for Food Safety and Applied Nutrition, U.S. Food and Drug Administration, College Park, MD, USA.

This protocol outlines the all the steps necessary to become a GenomeTrakr data contributor. GenomeTrakr is an international genomic reference database of mostly food and environmental isolates from foodborne pathogens. The data and analyses are housed at the National Center for Biotechnology Information (NCBI), which is a database freely available to anyone in the world. The Pathogen Detection browser at NCBI computes daily cluster results adding the newly submitted data to the existing phylogenetic clusters of closely related genomes. Contributors to this database can see how their new isolates are related to the real-time foodborne pathogen surveillance program established in the USA and a few other countries, and at the same time adding valuable new data to the reference database.
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http://dx.doi.org/10.1007/978-1-4939-9000-9_17DOI Listing
June 2019

Draft Genome Sequences of 57 Salmonella enterica Strains from Selected U.S. Swine Feed Mills.

Microbiol Resour Announc 2018 Nov 1;7(17). Epub 2018 Nov 1.

Food Science Institute, Kansas State University, Manhattan, Kansas, USA.

The number of Salmonella infection cases linked to pork products has increased. Pathogen presence in the feed mill environment is one of the many potential transmission routes into the food production chain. Here, we describe the draft genome sequences of 57 Salmonella enterica isolates from selected U.S. swine feed mills.
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http://dx.doi.org/10.1128/MRA.01191-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6256493PMC
November 2018

In-depth comparative analysis of Illumina MiSeq run metrics: Development of a wet-lab quality assessment tool.

Mol Ecol Resour 2019 Mar 17;19(2):377-387. Epub 2019 Jan 17.

Department of Microbiology, Center for Food Safety and Applied Nutrition, US Food and Drug Administration, College Park, Maryland.

Whole genome sequencing of bacterial isolates has become a daily task in many laboratories, generating incredible amounts of data. However, data acquisition is not an end in itself; the goal is to acquire high-quality data useful for understanding genetic relationships. Having a method that could rapidly determine which of the many available run metrics are the most important indicators of overall run quality and having a way to monitor these during a given sequencing run would be extremely helpful to this effect. Therefore, we compared various run metrics across 486 MiSeq runs, from five different machines. By performing a statistical analysis using principal components analysis and a K-means clustering algorithm of the metrics, we were able to validate metric comparisons among instruments, allowing for the development of a predictive algorithm, which permits one to observe whether a given MiSeq run has performed adequately. This algorithm is available in an Excel spreadsheet: that is, MiSeq Instrument & Run (In-Run) Forecast. Our tool can help verify that the quantity/quality of the generated sequencing data consistently meets or exceeds recommended manufacturer expectations. Patterns of deviation from those expectations can be used to assess potential run problems and plan preventative maintenance, which can save valuable time and funding resources.
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http://dx.doi.org/10.1111/1755-0998.12973DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6487961PMC
March 2019

Salmonella enterica Phylogeny Based on Whole-Genome Sequencing Reveals Two New Clades and Novel Patterns of Horizontally Acquired Genetic Elements.

mBio 2018 11 27;9(6). Epub 2018 Nov 27.

Center for Food Safety & Applied Nutrition, U.S. Food & Drug Administration, College Park, Maryland, USA

Using whole-genome sequence (WGS) data from the GenomeTrakr network, a globally distributed network of laboratories sequencing foodborne pathogens, we present a new phylogeny of comprising 445 isolates from 266 distinct serovars and originating from 52 countries. This phylogeny includes two previously unidentified subsp. clades. Serovar Typhi is shown to be nested within clade A. Our findings are supported by both phylogenetic support, based on a core genome alignment, and Bayesian approaches, based on single-nucleotide polymorphisms. Serovar assignments were refined by analysis using SeqSero. More than 10% of serovars were either polyphyletic or paraphyletic. We found variable genetic content in these isolates relating to gene mobilization and virulence factors which have different distributions within clades. Gifsy-1- and Gifsy-2-like phages appear more prevalent in clade A; other viruses are more evenly distributed. Our analyses reveal IncFII is the predominant plasmid replicon in Few core or clade-defining virulence genes are observed, and their distributions appear probabilistic in nature. Together, these patterns demonstrate that genetic exchange within is more extensive and frequent than previously realized, which significantly alters how we view the genetic structure of the bacterial species. Rapid improvements in nucleotide sequencing access and affordability have led to a drastic increase in availability of genetic information. This information will improve the accuracy of molecular descriptions, including serovars, within Although the concept of serovars continues to be useful, it may have more significant limitations than previously understood. Furthermore, the discrete absence or presence of specific genes can be an unstable indicator of phylogenetic identity. Whole-genome sequencing provides more rigorous tools for assessing the distributions of these genes. Our phylogenetic and genetic content analyses reveal how active genetic elements are dynamically distributed within a species, allowing us to better understand genetic reservoirs and underlying bacterial evolution.
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http://dx.doi.org/10.1128/mBio.02303-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6282209PMC
November 2018

Genomics of Salmonella contaminating backyard production systems reveals persistence and transmission of genetically related Salmonella on a farm basis.

Zoonoses Public Health 2018 12 27;65(8):1008-1014. Epub 2018 Sep 27.

Escuela de Medicina Veterinaria, Facultad de Ciencias de la Vida, Universidad Andres Bello, Santiago, Chile.

Animals raised in backyard productive systems (BPS) have been frequently associated with Salmonella outbreaks. Several serovars have caused these events, showing that different BPSs can be contaminated by distinct Salmonella serovars. The aim of this study was to characterize the genomic diversity of Salmonella isolates obtained from BPSs in Central Chile to understand their genomic relatedness. A whole-genome SNP-based phylogenetic analysis of 22 Salmonella isolates from 12 locations revealed that S. Typhimurium isolates clustered based on the BPS that they were originally isolated from, and the same was established for S. Enteritidis isolates. Furthermore, our genomic analysis shows that animals from different species (i.e., a chicken, a duck and a pig) carried genetically related S. Typhimurium strains within the same BPS. Moreover, some of these genetically related isolates were obtained in different years (2013 and 2014), indicating that farm-specific Salmonella can persist in BPSs for multiple years and that interspecies transmission is plausible in this environment. Understanding the dynamics of interspecies transmission of Salmonella serovars within a contaminated BPS is fundamental to the design of mitigation strategies to reduce outbreaks of human Salmonella associated with backyard production systems.
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http://dx.doi.org/10.1111/zph.12526DOI Listing
December 2018

Genes significantly associated with lineage II food isolates of Listeria monocytogenes.

BMC Genomics 2018 Sep 25;19(1):708. Epub 2018 Sep 25.

Division of Microbiology, Office of Regulatory Science, Center for Food Safety and Applied Nutrition, U.S. Food and Drug Administration, College Park, MD, USA.

Background: Listeria monocytogenes is a widespread foodborne pathogen that can cause listeriosis, a potentially fatal infection. L. monocytogenes is subdivided into four phylogenetic lineages, with the highest incidence of listeriosis occurring within lineage I followed by lineage II. Strains of L. monocytogenes differ in their phenotypic characteristics, including virulence. However, the genetic bases for these observed differences are not well understood, and current efforts to monitor L. monocytogenes in food consider all strains to be equally virulent. We use a comparative genomics approach to identify genes and single nucleotide polymorphisms (SNPs) in 174 clinical and food isolates of L. monocytogenes that potentially contribute to virulence or the capacity to adapt to food environments.

Results: No SNPs are significantly associated with food or clinical isolates. No genes are significantly associated with food or clinical isolates from lineage I, but eight genes consisting of multiple homologues are associated with lineage II food isolates. These include three genes which encode hypothetical proteins, the cadmium resistance genes cadA and cadC, the multi-drug resistance gene ebrB, a quaternary ammonium compound resistance gene qac, and a regulatory gene. All eight genes are plasmid-borne, and most closed L. monocytogenes plasmids carry at least five of the genes (24/27). In addition, plasmids are more frequently associated with lineage II food isolates than with lineage II clinical isolates.

Conclusions: We identify eight genes that are significantly associated with food isolates in lineage II. Interestingly, the eight genes are virtually absent in lineage II outbreak isolates, are composed of homologues which show a nonrandom distribution among lineage I serotypes, and the sequences are highly conserved across 27 closed Listeria plasmids. The functions of these genes should be explored further and will contribute to our understanding of how L. monocytogenes adapts to the host and food environments. Moreover, these genes may also be useful as markers for risk assessment models of either pathogenicity or the ability to proliferate in food and the food processing environment.
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http://dx.doi.org/10.1186/s12864-018-5074-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6157050PMC
September 2018