In 2020, an outbreak of Salmonella Hadar illnesses was linked to contact with non-commercial, privately owned (backyard) poultry including live chickens, turkeys, and ducks, resulting in 848 illnesses. From late 2020 to 2021, this Salmonella Hadar strain caused an outbreak that was linked to ground turkey consumption. Core genome multilocus sequence typing (cgMLST) analysis determined that the Salmonella Hadar isolates detected during the outbreak linked to backyard poultry and the outbreak linked to ground turkey were closely related genetically (within 0-16 alleles). Epidemiological and traceback investigations were unable to determine how Salmonella Hadar detected in backyard poultry and ground turkey were linked, despite this genetic relatedness. Enhanced molecular characterization methods, such as analysis of the pangenome of Salmonella isolates, might be necessary to understand the relationship between these two outbreaks. Similarly, enhanced data collection during outbreak investigations and further research could potentially aid in determining whether these transmission vehicles are truly linked by a common source and what reservoirs exist across the poultry industries that allow Salmonella Hadar to persist. Further work combining epidemiological data collection, more detailed traceback information, and genomic analysis tools will be important for monitoring and investigating future enteric disease outbreaks.

Whole genome sequencing is replacing traditional laboratory surveillance methods as the primary tool to track and characterize clusters and outbreaks of the foodborne and zoonotic pathogen Salmonella enterica (S. enterica). In this study, 438 S. enterica isolates representing 35 serovars and 13 broad vehicle categories from one hundred epidemiologically confirmed outbreaks were evaluated for genetic variation to develop epidemiologically relevant interpretation guidelines for Salmonella disease cluster detection. The Illumina sequences were analyzed by core genome multi-locus sequence typing (cgMLST) and screened for antimicrobial resistance (AR) determinants and plasmids. Ninety-three of the one hundred outbreaks exhibited a close allele range (less than 10 allele differences with a subset closer than 5). The remaining seven outbreaks showed increased variation, of which three were considered polyclonal. A total of 16 and 28 outbreaks, respectively, showed variations in the AR and plasmid profiles. The serovars Newport and I 4,[5],12:i:-, as well as the zoonotic and poultry product vehicles, were overrepresented among the outbreaks, showing increased variation. A close allele range in cgMLST profiles can be considered a reliable proxy for epidemiological relatedness for the vast majority of S. enterica outbreak investigations. Variations associated with mobile elements happen relatively frequently during outbreaks and could be reflective of changing selective pressures.

Bacterial relatedness measured using select chromosomal loci forms the basis of public health genomic surveillance. While approximating vertical evolution through this approach has proven exceptionally valuable for understanding pathogen dynamics, it excludes a fundamental dimension of bacterial evolution-horizontal gene transfer. Incorporating the accessory genome is the logical remediation and has recently shown promise in expanding epidemiological resolution for enteric pathogens. Employing k-mer-based Jaccard index analysis, and a novel genome length distance metric, we computed pangenome (i.e., core and accessory) relatedness for the globally important pathogen Salmonella enterica serotype Typhi (Typhi), and graphically express both vertical (homology-by-descent) and horizontal (homology-by-admixture) evolutionary relationships in a reticulate network of over 2,200 U.S. Typhi genomes. This analysis revealed non-random structure in the Typhi pangenome that is driven predominantly by the gain and loss of mobile genetic elements, confirming and expanding upon known epidemiological patterns, revealing novel plasmid dynamics, and identifying avenues for further genomic epidemiological exploration. With an eye to public health application, this work adds important biological context to the rapidly improving ways of analyzing bacterial genetic data and demonstrates the value of the accessory genome to infer pathogen epidemiology and evolution.IMPORTANCEGiven bacterial evolution occurs in both vertical and horizontal dimensions, inclusion of both core and accessory genetic material (i.e., the pangenome) is a logical step toward a more thorough understanding of pathogen dynamics. With an eye to public, and indeed, global health relevance, we couple contemporary tools for genomic analysis with decades of research on mobile genetic elements to demonstrate the value of the pangenome, known and unknown, annotated, and hypothetical, for stratification of Salmonella enterica serovar Typhi (Typhi) populations. We confirm and expand upon what is known about Typhi epidemiology, plasmids, and antimicrobial resistance dynamics, and offer new avenues of exploration to further deduce Typhi ecology and evolution, and ultimately to reduce the incidence of human disease.

Background:The Global Typhoid Genomics Consortium was established to bring together the typhoid research community to aggregate and analyse Salmonella enterica serovar Typhi (Typhi) genomic data to inform public health action. This analysis, which marks 22 years since the publication of the first Typhi genome, represents the largest Typhi genome sequence collection to date (n=13,000). Methods:This is a meta-analysis of global genotype and antimicrobial resistance (AMR) determinants extracted from previously sequenced genome data and analysed using consistent methods implemented in open analysis platforms GenoTyphi and Pathogenwatch. Results:Compared with previous global snapshots, the data highlight that genotype 4.3.1 (H58) has not spread beyond Asia and Eastern/Southern Africa; in other regions, distinct genotypes dominate and have independently evolved AMR. Data gaps remain in many parts of the world, and we show the potential of travel-associated sequences to provide informal 'sentinel' surveillance for such locations. The data indicate that ciprofloxacin non-susceptibility (>1 resistance determinant) is widespread across geographies and genotypes, with high-level ciprofloxacin resistance (≥3 determinants) reaching 20% prevalence in South Asia. Extensively drug-resistant (XDR) typhoid has become dominant in Pakistan (70% in 2020) but has not yet become established elsewhere. Ceftriaxone resistance has emerged in eight non-XDR genotypes, including a ciprofloxacin-resistant lineage (4.3.1.2.1) in India. Azithromycin resistance mutations were detected at low prevalence in South Asia, including in two common ciprofloxacin-resistant genotypes. Conclusions:The consortium's aim is to encourage continued data sharing and collaboration to monitor the emergence and global spread of AMR Typhi, and to inform decision-making around the introduction of typhoid conjugate vaccines (TCVs) and other prevention and control strategies. Funding:No specific funding was awarded for this meta-analysis. Coordinators were supported by fellowships from the European Union (ZAD received funding from the European Union's Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie grant agreement No 845681), the Wellcome Trust (SB, Wellcome Trust Senior Fellowship), and the National Health and Medical Research Council (DJI is supported by an NHMRC Investigator Grant [GNT1195210]).

The dissemination of antimicrobial-resistant genes in Gram-negative bacteria represents a global threat to healthcare systems. It is already well documented that multidrug resistance E. coli and Salmonella are of great importance for their lack of response to antibiotics commonly used to treat infections. This study aims to identify antimicrobial resistance markers found in E. coli and Salmonella strains isolated from beef, swine, and poultry samples collected in the Dominican Republic. Isolates (n=384) were analyzed using Whole Genome Sequencing (WGS). The isolates (n=384) were analyzed using Whole Genome Sequencing (WGS). Confirmed E. coli (n=26) and Salmonella (n=72) genome De novo assemblies were screened for resistance determinants and plasmid replicons. The Analysis revealed that the most frequent genes found in E. coli isolates were blaTEM-1B [60.0% (15/25)] and the tet(A) encoding for tetracycline efflux pump [53.8% (14/25)]. For Salmonella, [34.7% (25/72)] of the isolates were multidrug-resistant Salmonella entericaserovar Infantis harboring the blaCTX-M-65 gene. These isolates also carried aac(3)-IV, aph(4)-Ia, ant(3'')-Ia, and aph(3')-Ia, genes, associated with resistance to aminoglycosides class, chromosomal point mutations gyrA(D87Y) and parC(T57S), tet(A), and floR genes. The plasmid replicon most closely related to these E. coli and Salmonella was Col(pHAD28) at 48.0% (12/25) and 35.2% (25/72), respectively. In general, isolates showed high genotypic consistency between serotypes and sequence types (STs). There is limited information on antimicrobial susceptibility patterns associated with foodborne pathogens found in the Dominican Republic. This study provides insight into the spread of antimicrobial resistance genes in E. coli and Salmonella in the Dominican Republic.

AbstractPublic health genomic surveillance systems typically measure genome relatedness and infer molecular epidemiological relationships using chromosomal loci alone – an approximation of vertical evolution, or homology-by-descent. The accessory genome, composed of plasmids and other mobile genetic elements, reflects horizontal gene transfer and serves as an important mechanism of bacterial evolution, enabling rapid adaptation. Measuring homology in the accessory genome – homology-by-admixture – could offer important molecular epidemiological information for public health application. We applied Jaccard Index and a novel genome length distance metric to compute pangenome relatedness for the globally-important pathogenSalmonella entericaserotype Typhi (Typhi), and graphically express both homology-by-descent and homology-by-admixture in a reticulate network. Jaccard Index Network Analysis revealed structure in the Typhi pangenome that can be harnessed to enhance discriminatory power for surveillance, track antimicrobial resistance, and refine our understanding of homology for outbreak management and prevention. This offers a more intricate, multidimensional framework for understanding pathogen evolution.Significance StatementBacterial relatedness is often measured and visualized using chromosomal comparison and phylogenetic trees. While valuable, this approach captures only the vertical evolutionary dimension and excludes genetic material acquired or lost through horizontal gene transfer. We present an approach for measuring and visualizing bacterial relatedness using all core and accessory genetic material and discuss the interpretation of resulting reticulate networks of bacterial genomes. In application toSalmonellaTyphi, Jaccard Index Network Analysis revealed structure in populations of this pathogen that may be harnessed for public health applications. This approach captures both vertical and horizontal evolutionary dimensions, offering an intricate genetic framework for exploring pathogen evolution.

Genomic characterization of an Escherichia coli O157:H7 strain linked to leafy greens-associated outbreaks dates its emergence to late 2015. One clade has notable ac-cessory genomic content and a previously described mu-tation putatively associated with increased arsenic toler-ance. This strain is a reoccurring, emerging, or persistent strain causing illness over an extended period.

Public health genomic surveillance systems typically measure genome relatedness and infer molecular epidemiological relationships using chromosomal loci alone – an approximation of vertical evolution, or homology-by-descent. The accessory genome, composed of plasmids and other mobile genetic elements, reflects horizontal gene transfer and serves as an important mechanism of bacterial evolution, enabling rapid adaptation. Measuring homology in the accessory genome – homology-by-admixture – could offer important molecular epidemiological information for public health application. We applied Jaccard Index and a novel genome length distance metric to compute pangenome relatedness for the globally-important pathogen Salmonella enterica serotype Typhi (Typhi), and graphically express both homology-by-descent and homology-by-admixture in a reticulate network. Jaccard Index Network Analysis revealed structure in the Typhi pangenome that can be harnessed to enhance discriminatory power for surveillance, track antimicrobial resistance, and refine our understanding of homology for outbreak management and prevention. This offers a more intricate, multidimensional framework for understanding pathogen evolution.Significance Statement Bacterial relatedness is often measured and visualized using chromosomal comparison and phylogenetic trees. While valuable, this approach captures only the vertical evolutionary dimension and excludes genetic material acquired or lost through horizontal gene transfer. We present an approach for measuring and visualizing bacterial relatedness using all core and accessory genetic material and discuss the interpretation of resulting reticulate networks of bacterial genomes. In application to Salmonella Typhi, Jaccard Index Network Analysis revealed structure in populations of this pathogen that may be harnessed for public health applications. This approach captures both vertical and horizontal evolutionary dimensions, offering an intricate genetic framework for exploring pathogen evolution.### Competing Interest StatementThe authors have declared no competing interest.

Colistin is a last-resort antibiotic used to treat infections caused by multidrug-resistant Gram-negative bacteria. People with a history of travel to the Dominican Republic have become sick with pathogenic bacteria carrying the mobile colistin resistance gene, mcr-1, during and after traveling. This investigation aimed to identify mcr genes in Enterobacteriaceae isolated from food animal sources in the Dominican Republic. Three hundred and eleven samples were tested, from which 1354 bacterial isolates were obtained. Real-time PCR tests showed that 70.7% (220 out of 311) of the samples and 3.2% (44 out of 1354) of the isolates tested positive for the mcr gene. All RT-PCR presumptive mcr-positive isolates (n = 44) and a subset (n = 133) of RT-PCR presumptive mcr-negative isolates were subjected to whole-genome sequencing. WGS analysis showed that 39 isolates carried the mcr gene, with 37 confirmed as positive through RT-PCR and two as negative. Further, all of the mcr-positive genomes were identified as Escherichia coli and all contained a IncX4 plasmid replicon. Resistant determinants for other antibiotics important for human health were found in almost all isolates carrying mcr genes.

In the United States, reports of Salmonella enterica carrying mcr-1 remain rare in humans, but when observed, the infection is often associated with travel. Here, we report 14 mcr-1-positive Salmonella enterica isolates from patients in the United States that reported travel to the Dominican Republic within the 12 months before illness.

Resistance genes encoding β-lactamases (BLs) confer resistance to the widely prescribed antibiotic class β-lactams. Therefore, it is important to assess the prevalence of BL genes in clinical or environmental samples for monitoring the spreading of these genes into pathogens and estimating public health risk.

From a global view of antimicrobial resistance over different sectors, seafood and the marine environment are often considered as potential reservoirs of antimicrobial resistance genes (ARGs) and mobile genetic elements (MGEs); however, there are few studies and sparse results on this sector. This study aims to provide new data and insights regarding the content of resistance markers in various seafood samples and sources, and therefore the potential exposure to humans in a global One Health approach. An innovative high throughput qPCR screening was developed and validated in order to simultaneously investigate the presence of 41 ARGs and 33 MGEs including plasmid replicons, integrons, and insertion sequences in Gram-negative bacteria. Analysis of 268 seafood isolates from the bacterial microflora of cod (n = 24), shellfish (n = 66), flat fishes (n = 53), shrimp (n = 10), and horse mackerel (n = 115) show the occurrence of sul-1, ant(3″)-Ia, aph(3′)-Ia, strA, strB, dfrA1, qnrA, and blaCTX-M-9 genes in Pseudomonas spp., Providencia spp., Klebsiella spp., Proteus spp., and Shewanella spp. isolates and the presence of MGEs in all bacterial species investigated. We found that the occurrence of MGE may be associated with the seafood type and the environmental, farming, and harvest conditions. Moreover, even if MGE were detected in half of the seafood isolates investigated, association with ARG was only identified for twelve isolates. The results corroborate the hypothesis that the incidence of antimicrobial-resistant bacteria (ARB) and ARG decreases with increasing distance from potential sources of fecal contamination. This unique and original high throughput micro-array designed for the screening of ARG and MGE in Gram-negative bacteria could be easily implementable for monitoring antimicrobial resistance gene markers in diverse contexts.

Despite being linked to a number of recent poultry-associated outbreaks in the United States, few reference genomes are available for Salmonella enterica serotype Hadar. Here, we address this need by reporting 18 Salmonella Hadar genomes from samples collected from patients in the United States between 2014 and 2020.

Between 2018 and 2019, Salmonella enterica serotype Reading caused a large, multistate outbreak linked to contact with raw turkey products in the United States. Here, we provide five Salmonella Reading reference genomes collected from US patients between 2016 and 2018.

Abstract Background Typhoid fever, caused by Salmonella Typhi, is fatal in 12%–30% of patients not treated with appropriate antibiotics. In 2016, a large outbreak of extensively drug-resistant (XDR) Typhi infections began in Pakistan with cases reported globally, including the United States. In 2021, the Centers for Disease Control and Prevention (CDC) issued a health advisory on XDR infections among U.S. residents without international travel. We describe resistance of Typhi infections diagnosed in the United States to help guide treatment decisions. Methods Typhoid fever is a nationally notifiable disease. Health departments report cases to CDC through the National Typhoid and Paratyphoid Fever Surveillance system. Isolates are submitted to the National Antimicrobial Resistance Monitoring System for antimicrobial susceptibility testing (AST) using broth microdilution. AST results are categorized by Clinical and Laboratory Standards Institute criteria. We defined XDR as resistant to ceftriaxone, ampicillin, chloramphenicol, and co-trimoxazole, and nonsusceptible to ciprofloxacin. Results During 2008–2019, of 4,637 Typhi isolates, 52 (1%) were ceftriaxone resistant (axo-R); 71% were ciprofloxacin nonsusceptible, 1 azithromycin resistant (azm-R), and none meropenem resistant. XDR was first detected in 2018, in 2% of 474 isolates and increased to 7% of 535 in 2019. Of the 52 axo-R isolates, 46 were XDR, of which 45 were from travelers to Pakistan, and one from a non-traveler; 6 were not XDR, of which 4 were linked to travel to Iraq. In preliminary 2020 reports, 23 isolates were XDR; 14 were from travelers to Pakistan, 8 from non-travelers, and 1 from someone with unknown travel status. Among those with XDR infection, median age was 11 years (range 1–62), 54% were female, and 62% were from 6 states. Conclusion Ceftriaxone-resistant Typhi infections, mostly XDR, are increasing. Clinicians should ask patients with suspected Typhi infections about travel and adjust treatment based on susceptibility results. Carbapenem, azithromycin, or both may be considered for empiric therapy of typhoid fever among travelers to Pakistan or Iraq and in uncommon instances when persons report no international travel. Ceftriaxone is an empiric therapy option for travelers to countries other than Pakistan and Iraq. Disclosures All Authors: No reported disclosures

Background Plasmids are mobile genetic elements, key in the dissemination of antibiotic resistance, virulence determinants and other adaptive traits in bacteria. Obtaining a robust method for plasmid classification is necessary to better understand the genetics and epidemiology of many pathogens. Until now, plasmid classification systems focused on specific traits, which limited their precision and universality. The definition of plasmid taxonomic units (PTUs), based on average nucleotide identity metrics, allows the generation of a universal plasmid classification scheme, applicable to all bacterial taxa. Here we present COPLA, a software able to assign plasmids to known and novel PTUs, based on their genomic sequence. Results We implemented an automated pipeline able to assign a given plasmid DNA sequence to its cognate PTU, and assessed its performance using a sample of 1000 unclassified plasmids. Overall, 41% of the samples could be assigned to a previously defined PTU, a number that reached 63% in well-known taxa such as the Enterobacterales order. The remaining plasmids represent novel PTUs, indicating that a large fraction of plasmid backbones is still uncharacterized. Conclusions COPLA is a bioinformatic tool for universal, species-independent, plasmid classification. Offered both as an automatable pipeline and an open web service, COPLA will help bacterial geneticists and clinical microbiologists to quickly classify plasmids.

Additional file 1. List of reference plasmids. A complete list of the plasmids from release 84 of the NCBI RefSeq Plasmid database used to build the reference network and its assignation to PTUs, the current COPLA reference database.

The Plasmid Taxonomic Unit (PTU) concept is an initial step for a natural classification of plasmids. Here we present COPLA, a software for plasmid assignation to existing PTUs. To assess its performance, we used a sample of 1,000 plasmids missing from its current database. Overall, 41% of samples could be assigned an existing PTU (63% within the most abundant order, Enterobacterales ), while 4% of samples could help to define new PTUs once COPLA database was updated.

Abstract Background Colistin, once seldom used clinically, has resurged as a “last resort antibiotic” for multidrug-resistant infections and is still used in animal agriculture in countries outside the United States. During 2015–2018, 8 plasmid-mediated, mobile colistin resistance genes (mcr-1 to mcr-8) were each found in one or more clinical, animal, food, and environmental bacterial sources. We describe the epidemiology of mcr genes in enteric pathogens from US patients. Methods State public health laboratories have performed whole-genome sequencing on enteric bacterial pathogens since 2015, and some have sequenced older isolates. We screened sequences of isolates collected through 2019 for mcr genes using a workflow based on ResFinder 3.0. State health officials interviewed patients for clinical and epidemiologic information, including demographics, hospitalization, and travel history. Results We identified 41 patient isolates with mcr genes collected from stool, urine, and blood during 2008–2019. These included 37 nontyphoidal Salmonella (31 mcr-1, 6 mcr-3), 2 Vibrio (both mcr-4), and 2 Shiga toxin-producing E. coli (both mcr-1). The median patient age was 34 years (interquartile range: 24–54) and 54% were female. Of 23 patients with comorbidity data, 2 (9%) had immunodeficiency, 2 (9%) had past abdominal surgeries, and 1 (4%) had cancer. Patients sought care at doctor’s offices (46%), emergency rooms (35%), and urgent care clinics (19%); 24% were hospitalized for the enteric illness. None died. Among 36 with information, 35 (97%) travelled internationally in the 12 months before illness; 30 (94%) of 32 traveled in the 7 days before. Only 4 (15%) of 27 had contact with a healthcare setting during their trip; common destinations were the Dominican Republic (35%), Vietnam (24%), Thailand (15%), and China (12%). Conclusion The data strongly suggest that many patients acquired infection abroad. Nearly one in four were hospitalized, raising concerns that plasmids carrying mcr genes could spread among patients hospitalized with infections caused by multidrug-resistant pathogens for which colistin is the only available treatment. The acquisition of mcr genes by US travelers highlights the need for a global approach to antimicrobial stewardship. Disclosures All Authors: No reported disclosures

Abstract Background Multidrug-resistant (MDR) Shigella sonnei infections are a serious public health threat, and outbreaks are common among men who have sex with men (MSM). In February 2020, Australia’s Department of Health notified CDC of extensively drug-resistant (XDR) S. sonnei in 2 Australian residents linked to a cruise that departed from Florida. We describe an international outbreak of XDR S. sonnei and report on trends in MDR among S. sonnei in the United States. Methods Health departments (HDs) submit every 20th Shigella isolate to CDC’s National Antimicrobial Resistance Monitoring System (NARMS) laboratory for susceptibility testing. We defined MDR as decreased susceptibility to azithromycin (MIC ≥32 µg/mL) with resistance to ampicillin, ciprofloxacin, and cotrimoxazole, and XDR as MDR with additional resistance to ceftriaxone. We used PulseNet, the national subtyping network for enteric disease surveillance, to identify US isolates related to the Australian XDR isolates by short-read whole genome sequencing. We screened these isolates for resistance determinants (ResFinder v3.0) and plasmid replicons (PlasmidFinder) and obtained patient histories from HDs. We used long-read sequencing to generate closed plasmid sequences for 2 XDR isolates. Results NARMS tested 2,781 S. sonnei surveillance isolates during 2011–2018; 80 (2.9%) were MDR, including 1 (0.04%) that was XDR. MDR isolates were from men (87%), women (9%), and children (4%). MDR increased from 0% in 2011 to 15.3% in 2018 (Figure). In 2020, we identified XDR isolates from 3 US residents on the same cruise as the Australians. The US residents were 41–42 year-old men; 2 with available information were MSM. The US and Australian isolates were highly related (0–1 alleles). Short-read sequence data from all 3 US isolates mapped to the blaCTX-M-27 harboring IncFII plasmids from the 2 Australian isolates with >99% nucleotide identity. blaCTX-M-27 genes confer ceftriaxone resistance. Increase in Percentage of Shigella sonnei Isolates with Multidrug Resistance* in the United States, 2011–2018† Conclusion MDR S. sonnei is increasing and is most often identified among men. XDR S. sonnei infections are emerging and are resistant to all recommended antibiotics, making them difficult to treat without IV antibiotics. This outbreak illustrates the alarming capacity for XDR S. sonnei to disseminate globally among at-risk populations, such as MSM. Disclosures All Authors: No reported disclosures